Combined use of GDF traps and erythropoietin receptor activators to increase red blood cell levels

ABSTRACT

In certain aspects, the present invention provides compositions and methods for increasing red blood cell and/or hemoglobin levels in vertebrates, including rodents and primates, and particularly in humans.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/157,261, (now U.S. Pat. No. 10,689,427), which is a continuation ofU.S. application Ser. No. 15/790,400 (now U.S. Pat. No. 10,131,700),which is a continuation of U.S. application Ser. No. 15/221,341, filedJul. 27, 2016 (now U.S. Pat. No. 9,932,379), which is a continuation ofU.S. application Ser. No. 14/201,192, filed Mar. 7, 2014 (now U.S. Pat.No. 9,439,945), which is a continuation of U.S. application Ser. No.13/542,269, filed Jul. 5, 2012 (now U.S. Pat. No. 8,703,927), which is acontinuation of U.S. application Ser. No. 12/856,420, filed Aug. 13,2010 (now U.S. Pat. No. 8,216,997), which is a continuation-in-part ofU.S. application Ser. No. 12/583,177, filed Aug. 13, 2009 (now U.S. Pat.No. 8,058,229) and International Application Serial No.PCT/US2009/004659, filed on Aug. 13, 2009. U.S. application Ser. No.12/856,420 claims the benefit of priority from U.S. ProvisionalApplication No. 61/305,901, filed Feb. 18, 2010. U.S. application Ser.No. 12/583,177 claims the benefit of priority of U.S. ProvisionalApplication 61/189,094, filed Aug. 14, 2008. The specifications of eachof the foregoing applications are incorporated herein by reference intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Feb. 19, 2020, is named1848179-0002-107-107_SL.TXT, and is 71,308 bytes in size.

BACKGROUND OF THE INVENTION

The mature red blood cell, or erythrocyte, is responsible for oxygentransport in the circulatory systems of vertebrates. Red blood cellscontain high concentrations of hemoglobin, a protein that binds oxygenin the lungs at relatively high partial pressure of oxygen (pO₂) anddelivers oxygen to areas of the body with a relatively low pO₂.

Mature red blood cells are produced from pluripotent hematopoietic stemcells in a process termed erythropoiesis. Postnatal erythropoiesisoccurs primarily in the bone marrow and in the red pulp of the spleen.The coordinated action of various signaling pathways control the balanceof cell proliferation, differentiation, survival and death. Under normalconditions, red blood cells are produced at a rate that maintains aconstant red cell mass in the body, and production may increase ordecrease in response to various stimuli, including increased ordecreased oxygen tension or tissue demand. The process of erythropoiesisbegins with the formation of lineage committed precursor cells andproceeds through a series of distinct precursor cell types. The finalstages of erythropoiesis occur as reticulocytes are released into thebloodstream and lose their mitochondria and ribosomes while assuming themorphology of mature red blood cell. An elevated level of reticulocytes,or an elevated reticulocyte:erythrocyte ratio, in the blood isindicative of increased red blood cell production rates.

Erythropoietin (EPO) is widely recognized as the most significantpositive regulator of postnatal erythropoiesis in vertebrates. EPOregulates the compensatory erythropoietic response to reduced tissueoxygen tension (hypoxia) and low red blood cell levels or low hemoglobinlevels. In humans, elevated EPO levels promote red blood cell formationby stimulating the generation of erythroid progenitors in the bonemarrow and spleen. In the mouse, EPO enhances erythropoiesis primarilyin the spleen.

Effects of EPO are mediated by a cell-surface receptor belonging to thecytokine receptor superfamily. The human EPO receptor gene encodes a 483amino-acid transmembrane protein, whereas the active EPO receptor isthought to exist as a multimeric complex even in the absence of ligand(See U.S. Pat. No. 6,319,499). The cloned full-length EPO receptorexpressed in mammalian cells binds EPO with an affinity similar to thatof the native receptor on erythroid progenitor cells. Binding of EPO toits receptor causes a conformational change resulting in receptoractivation and biological effects including increased proliferation ofimmature erythroblasts, increased differentiation of immatureerythroblasts, and decreased apoptosis in erythroid progenitor cells(Liboi et al., 1993, Proc Natl Acad Sci USA 90:11351-11355; Koury etal., 1990, Science 248:378-381).

Various forms of recombinant EPO are used by physicians to increase redblood cell levels in a variety of clinical settings, and particularlyfor the treatment of anemia. Anemia is a broadly-defined conditioncharacterized by lower than normal levels of hemoglobin or red bloodcells in the blood. In some instances, anemia is caused by a primarydisorder in the production or survival of red blood cells. Morecommonly, anemia is secondary to diseases of other systems (Weatherall &Provan (2000) Lancet 355, 1169-1175). Anemia may result from a reducedrate of production or increased rate of destruction of red blood cellsor by loss of red blood cells due to bleeding. Anemia may result from avariety of disorders that include, for example, chronic renal failure,chemotherapy treatment, myelodysplastic syndrome, rheumatoid arthritis,and bone marrow transplantation.

Treatment with EPO typically causes a rise in hemoglobins by about 1-3g/dL in healthy humans over a period of weeks. When administered toanemic individuals, this treatment regimen often provides substantialincreases in hemoglobin and red blood cell levels and leads toimprovements in quality of life and prolonged survival. EPO is notuniformly effective, and many individuals are refractory to even highdoses (Horl et al. (2000) Nephrol Dial Transplant 15, 43-50). Over 50%of patients with cancer have an inadequate response to EPO,approximately 10% with end-stage renal disease are hyporesponsive(Glaspy et al. (1997) J Clin Oncol 15, 1218-1234; Demetri et al. (1998)J Clin Oncol 16, 3412-3425), and less than 10% with myelodysplasticsyndrome respond favorably (Estey (2003) Curr Opin Hematol 10, 60-67).Several factors, including inflammation, iron and vitamin deficiency,inadequate dialysis, aluminum toxicity, and hyperparathyroidism maypredict a poor therapeutic response. The molecular mechanisms ofresistance to EPO are as yet unclear. Recent evidence suggests thathigher doses of EPO may be associated with an increased risk ofcardiovascular morbidity, tumor growth, and mortality in some patientpopulations (Krapf et al., 2009, Clin J Am Soc Nephrol 4:470-480;Glaspy, 2009, Annu Rev Med 60:181-192). It has therefore beenrecommended that EPO-based therapeutic compounds(erythropoietin-stimulating agents, ESAs) be administered at the lowestdose sufficient to avoid the need for red blood cell transfusions(Jelkmann et al., 2008, Crit Rev Oncol. Hematol 67:39-61).

Thus, it is an object of the present disclosure to provide alternativemethods for increasing red blood cell levels in patients, which wouldpermit use of reduced doses of erythropoietin receptor activators.

SUMMARY OF THE INVENTION

In part, the disclosure demonstrates that GDF Traps may be used incombination (e.g., administered at the same time or different times, butgenerally in such a manner as to achieve overlapping pharmacologicaleffects) with EPO receptor activators to increase red blood cell levels(erythropoiesis) or treat anemia in patients in need thereof. In part,the disclosure demonstrates that a GDF Trap can be administered incombination with an EPO receptor activator to synergistically increaseformation of red blood cells in a patient. Thus, the effect of thiscombined treatment can be significantly greater than the sum of theeffects of the GDF Trap and the EPO receptor activator when administeredseparately at their respective doses. In certain embodiments, thissynergism may be advantageous since it enables target levels of redblood cells to be attained with lower doses of an EPO receptoractivator, thereby avoiding potential adverse effects or other problemsassociated with higher levels of EPO receptor activation.

An EPO receptor activator may stimulate erythropoiesis by directlycontacting and activating EPO receptor. In certain embodiments, the EPOreceptor activator is one of a class of compounds based on the 165amino-acid sequence of native EPO and generally known aserythropoiesis-stimulating agents (ESAs), examples of which are epoetinalfa, epoetin beta, epoetin delta, and epoetin omega. In otherembodiments, ESAs include synthetic EPO proteins (SEPs) and EPOderivatives with nonpeptidic modifications conferring desirablepharmacokinetic properties (lengthened circulating half-life), examplesof which are darbepoetin alfa and methoxy-polyethylene-glycol epoetinbeta. In certain embodiments, an EPO receptor activator may be an EPOreceptor agonist that does not incorporate the EPO polypeptide backboneor is not generally classified as an ESA. Such EPO receptor agonists mayinclude, but are not limited to, peptidic and nonpeptidic mimetics ofEPO, agonistic antibodies targeting EPO receptor, fusion proteinscomprising an EPO mimetic domain, and erythropoietin receptorextended-duration limited agonists (EREDLA).

In certain embodiments, an EPO receptor activator may stimulateerythropoiesis indirectly, without contacting EPO receptor itself, byenhancing production of endogenous EPO. For example, hypoxia-inducibletranscription factors (HIFs) are endogenous stimulators of EPO geneexpression that are suppressed (destabilized) under normoxic conditionsby cellular regulatory mechanisms. In part, the disclosure providesincreased erythropoiesis in a patient by combined treatment with a GDFTrap and an indirect EPO receptor activator with HIF stabilizingproperties, such as a prolyl hydroxylase inhibitor.

Variant ActRIIB polypeptides having a significantly decreased affinityfor activin (e.g., activin A and/or activin B) relative to other ActRIIBligands, such as GDF11 and/or myostatin, are referred to as GDF Traps.ActRIIB variants described herein are GDF Traps unless otherwise stated.In particular, the disclosure demonstrates that a GDF Trap which is asoluble form of ActRIIB polypeptide having an acidic residue at position79 of SEQ ID NO: 1, when administered in vivo, increases red blood celllevels in the blood. Therefore, in certain embodiments, the disclosureprovides methods for using GDF Traps to increase red blood cell andhemoglobin levels in patients and to treat disorders associated with lowred blood cell or hemoglobin levels in patients in need thereof. Asdescribed in U.S. patent application Ser. No. 12/012,652, incorporatedby reference herein, GDF Traps can be used to increase muscle mass anddecrease fat mass.

In certain aspects, the present disclosure provides GDF Traps that arevariant ActRIIB polypeptides, including ActRIIB polypeptides havingamino- and carboxy-terminal truncations and sequence alterations.Optionally, GDF Traps of the invention may be designed to preferentiallyantagonize one or more ligands of ActRIIB receptors, such as GDF8 (alsocalled myostatin), GDF11, Nodal, and BMP7 (also called OP-1). Examplesof GDF Traps include a set of variants derived from ActRIIB that havegreatly diminished affinity for activin. These variants exhibitdesirable effects on red blood cells while reducing effects on othertissues. Examples of such variants include those having an acidic aminoacid (e.g., aspartic acid, D, or glutamic acid, E) at the positioncorresponding to position 79 of SEQ ID NO.1. In certain embodiments, theGDF Trap polypeptide comprises an amino acid sequence that comprises,consists of, or consists essentially of, the amino acid sequence of SEQID NO: 7, 26, 28, 29, 32, 37 or 38, and polypeptides that are at least80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to any of the foregoing.

In certain aspects, the disclosure provides pharmaceutical preparationscomprising a GDF Trap that binds to an ActRIIB ligand such as GDF8,GDF11, activin (e.g., activin B), BMP7 or nodal, and a pharmaceuticallyacceptable carrier. Optionally, the GDF Trap binds to an ActRIIB ligandwith a Kd less than 10 micromolar, less than 1 micromolar, less than 100nanomolar, less than 10 nanomolar, or less than 1 nanomolar. Optionally,the GDF Trap inhibits ActRIIB signaling, such as intracellular signaltransduction events triggered by an ActRIIB ligand. A GDF Trap for usein such a preparation may be any of those disclosed herein, including,for example, GDF Traps having an amino acid sequence selected from SEQID NOs: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38 or 40, or GDF Traps havingan amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99%identical to an amino acid sequence selected from SEQ ID NOs: 2, 3, 7,11, 26, 28, 29, 32, 37, 38 or 40, or GDF Traps having an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to anamino acid sequence selected from SEQ ID NOs: 2, 3, 7, 11, 26, 28, 29,32, 37, 38 or 40 wherein the position corresponding to L79 in SEQ ID NO:1 is an acidic amino acid. A preferred GDF Trap for use in such apreparation consists of, or consists essentially of, the amino acidsequence of SEQ ID NO: 26. A GDF Trap may include a functional fragmentof a natural ActRIIB polypeptide, such as one comprising at least 10, 20or 30 amino acids of a sequence selected from SEQ ID NOs: 2, 3, 7, 11,26, 28, 29, 32, 37, 38 or 40 or a sequence of SEQ ID NO: 2, lacking theC-terminal 1, 2, 3, 4, 5 or 10 to 15 amino acids and lacking 1, 2, 3, 4or 5 amino acids at the N-terminus. A preferred polypeptide willcomprise a truncation relative to SEQ ID NO: 2 or 40 of between 2 and 5amino acids at the N-terminus and no more than 3 amino acids at theC-terminus. A GDF Trap may include one or more alterations in the aminoacid sequence of an ActRIIB polypeptide (e.g., in the ligand-bindingdomain) relative to a naturally occurring ActRIIB polypeptide. Thealteration in the amino acid sequence may, for example, alterglycosylation of the polypeptide when produced in a mammalian, insect orother eukaryotic cell or alter proteolytic cleavage of the polypeptiderelative to the naturally occurring ActRIIB polypeptide.

A GDF Trap may be a fusion protein that has, as one domain, an ActRIIBpolypeptide (e.g., a ligand-binding domain of an ActRIIB with one ormore sequence variations) and one or more additional domains thatprovide a desirable property, such as improved pharmacokinetics, easierpurification, targeting to particular tissues, etc. For example, adomain of a fusion protein may enhance one or more of in vivo stability,in vivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, multimerization of thefusion protein, and/or purification. GDF Trap fusion proteins mayinclude an immunoglobulin Fc domain (wild-type or mutant) or a serumalbumin. In certain embodiments, a GDF Trap fusion comprises arelatively unstructured linker positioned between the Fc domain and theextracellular ActRIIB domain. This unstructured linker may correspond tothe roughly 15 amino acid unstructured region at the C-terminal end ofthe extracellular domain of ActRIIB (the “tail”), or it may be anartificial sequence of between 3 and 5, 15, 20, 30, 50 or more aminoacids that are relatively free of secondary structure. A linker may berich in glycine and proline residues and may, for example, containrepeating sequences of threonine/serine and glycines (e.g., TG4 (SEQ IDNO: 13) or SG₄ (SEQ ID NO: 14) singlets or repeats) or a series of threeglycines. A fusion protein may include a purification subsequence, suchas an epitope tag, a FLAG tag, a polyhistidine sequence, and a GSTfusion. In certain embodiments, a GDF Trap fusion comprises a leadersequence. The leader sequence may be a native ActRIIB leader sequence ora heterologous leader sequence. In certain embodiments, the leadersequence is a Tissue Plasminogen Activator (TPA) leader sequence. In anembodiment, a GDF Trap fusion protein comprises an amino acid sequenceas set forth in the formula A-B-C. The B portion is an N- andC-terminally truncated ActRIIB polypeptide consisting of the amino acidsequence corresponding to amino acids 25-131 of SEQ ID NO: 2 or 40. TheA and C portions may be independently zero, one or more than one aminoacids, and both A and C portions are heterologous to B. The A and/or Cportions may be attached to the B portion via a linker sequence.

Optionally, a GDF Trap includes a variant ActRIIB polypeptide having oneor more modified amino acid residues selected from: a glycosylated aminoacid, a PEGylated amino acid, a farnesylated amino acid, an acetylatedamino acid, a biotinylated amino acid, an amino acid conjugated to alipid moiety, and an amino acid conjugated to an organic derivatizingagent. A pharmaceutical preparation may also include one or moreadditional compounds such as a compound that is used to treat anActRIIB-associated disorder. Preferably, a pharmaceutical preparation issubstantially pyrogen free. In general, it is preferable that a GDF Trapbe expressed in a mammalian cell line that mediates suitably naturalglycosylation of the GDF Trap so as to diminish the likelihood of anunfavorable immune response in a patient. Human and CHO cell lines havebeen used successfully, and it is expected that other common mammalianexpression vectors will be useful.

In certain aspects, the disclosure provides packaged pharmaceuticalscomprising a pharmaceutical preparation described herein and labeled foruse in increasing red blood cell levels in a human.

In certain aspects, the disclosure provides GDF Traps which are solubleActRIIB polypeptides comprising an altered ligand-binding (e.g.,GDF8-binding) domain. GDF Traps with altered ligand-binding domains maycomprise, for example, one or more mutations at amino acid residues suchas E37, E39, R40, K55, R56, Y60, A64, K74, W78, L79, D80, F82 and F101of human ActRIIB (numbering is relative to SEQ ID NO: 1). Optionally,the altered ligand-binding domain can have increased selectivity for aligand such as GDF8/GDF11 relative to a wild-type ligand-binding domainof an ActRIIB receptor. To illustrate, these mutations are demonstratedherein to increase the selectivity of the altered ligand-binding domainfor GDF11 (and therefore, presumably, GDF8) over activin: K74Y, K74F,K74I, L79D, L79E, and D80I. The following mutations have the reverseeffect, increasing the ratio of activin binding over GDF11: D54A, K55A,L79A and F82A. The overall (GDF11 and activin) binding activity can beincreased by inclusion of the “tail” region or, presumably, anunstructured linker region, and also by use of a K74A mutation. Othermutations that caused an overall decrease in ligand binding affinity,include: R40A, E37A, R56A, W78A, D80K, D80R, D80A, D80G, D80F, D80M andD80N. Mutations may be combined to achieve desired effects. For example,many of the mutations that affect the ratio of GDF11:Activin bindinghave an overall negative effect on ligand binding, and therefore, thesemay be combined with mutations that generally increase ligand binding toproduce an improved binding protein with ligand selectivity. In anexemplary embodiment, a GDF Trap is an ActRIIB polypeptide comprising anL79D or L79E mutation, optionally in combination with additional aminoacid substitutions, additions or deletions.

Optionally, a GDF Trap comprising an altered ligand-binding domain has aratio of K_(d) for activin binding to K_(d) for GDF8 binding that is atleast 2, 5, 10, or even 100 fold greater relative to the ratio for thewild-type ligand-binding domain. Optionally, the GDF Trap comprising analtered ligand-binding domain has a ratio of IC₅₀ for inhibiting activinto IC₅₀ for inhibiting GDF8/GDF11 that is at least 2, 5, 10, or even 100fold greater relative to the wild-type ActRIIB ligand-binding domain.Optionally, the GDF Trap comprising an altered ligand-binding domaininhibits GDF8/GDF11 with an IC₅₀ at least 2, 5, 10, or even 100 timesless than the IC₅₀ for inhibiting activin. These GDF Traps can be fusionproteins that include an immunoglobulin Fc domain (either wild-type ormutant). In certain cases, the subject soluble GDF Traps are antagonists(inhibitors) of GDF8 and/or GDF11.

Other GDF Traps are contemplated, such as the following. A GDF Trapfusion protein comprising a portion derived from the ActRIIB sequence ofSEQ ID NO: 1 or 39 and a second polypeptide portion, wherein the portionderived from ActRIIB corresponds to a sequence beginning at any of aminoacids 21-29 of SEQ ID NO: 1 or 39 (optionally beginning at 22-25 of SEQID NO: 1 or 39) and ending at any of amino acids 109-134 of SEQ ID NO: 1or 39, and wherein the GDF Trap fusion protein inhibits signaling byactivin, myostatin and/or GDF11 in a cell-based assay. The GDF Trapfusion protein above, wherein the portion derived from ActRIIBcorresponds to a sequence beginning at any of amino acids 20-29 of SEQID NO: 1 or 39 (optionally beginning at 22-25 of SEQ ID NO: 1 or 39) andending at any of amino acids 109-133 of SEQ ID NO: 1 or 39. The GDF Trapfusion protein above, wherein the portion derived from ActRIIBcorresponds to a sequence beginning at any of amino acids 20-24 of SEQID NO: 1 or 39 (optionally beginning at 22-25 of SEQ ID NO: 1 or 39) andending at any of amino acids 109-133 of SEQ ID NO: 1 or 39. The GDF Trapfusion protein above, wherein the portion derived from ActRIIBcorresponds to a sequence beginning at any of amino acids 21-24 of SEQID NO: 1 or 39 and ending at any of amino acids 109-134 of SEQ ID NO: 1or 39. The GDF Trap fusion protein above, wherein the portion derivedfrom ActRIIB corresponds to a sequence beginning at any of amino acids20-24 of SEQ ID NO: 1 or 39 and ending at any of amino acids 118-133 ofSEQ ID NO: 1 or 39. The GDF Trap fusion protein above, wherein theportion derived from ActRIIB corresponds to a sequence beginning at anyof amino acids 21-24 of SEQ ID NO: 1 or 39 and ending at any of aminoacids 118-134 of SEQ ID NO: 1 or 39. The GDF Trap fusion protein above,wherein the portion derived from ActRIIB corresponds to a sequencebeginning at any of amino acids 20-24 of SEQ ID NO: 1 or 39 and endingat any of amino acids 128-133 of SEQ ID NO: 1 or 39. The GDF Trap fusionprotein above, wherein the portion derived from ActRIIB corresponds to asequence beginning at any of amino acids 20-24 of SEQ ID NO: 1 or 39 andending at any of amino acids 128-133 of SEQ ID NO: 1 or 39. The GDF Trapfusion protein above, wherein the portion derived from ActRIIBcorresponds to a sequence beginning at any of amino acids 21-29 of SEQID NO: 1 or 39 and ending at any of amino acids 118-134 of SEQ ID NO: 1or 39. The GDF Trap fusion protein above, wherein the portion derivedfrom ActRIIB corresponds to a sequence beginning at any of amino acids20-29 of SEQ ID NO: 1 or 39 and ending at any of amino acids 118-133 ofSEQ ID NO: 1 or 39. The GDF Trap fusion protein above, wherein theportion derived from ActRIIB corresponds to a sequence beginning at anyof amino acids 21-29 of SEQ ID NO: 1 or 39 and ending at any of aminoacids 128-134 of SEQ ID NO: 1 or 39. The GDF Trap fusion protein above,wherein the portion derived from ActRIIB corresponds to a sequencebeginning at any of amino acids 20-29 of SEQ ID NO: 1 and ending at anyof amino acids 128-133 of SEQ ID NO: 1 or 39. Surprisingly, constructsbeginning at 22-25 of SEQ ID NO: 1 or 39 have activity levels greaterthan proteins having the full extracellular domain of human ActRIIB In apreferred embodiment, the GDF Trap fusion protein comprises, consistsessentially of, or consists of, an amino acid sequence beginning atamino acid position 25 of SEQ ID NO: 1 or 39 and ending at amino acidposition 131 of SEQ ID NO: 1 or 39. In another preferred embodiments,the GDF Trap polypeptide consists of, or consists essentially of, theamino acid sequence of SEQ ID NO: 7, 26, 28, 29, 32, 37 or 38. Any ofthe above GDF Trap fusion proteins may be produced as a homodimer. Anyof the above GDF Trap fusion proteins may have a heterologous portionthat comprises a constant region from an IgG heavy chain, such as an Fcdomain. Any of the above GDF Trap fusion proteins may comprise an acidicamino acid at the position corresponding to position 79 of SEQ ID NO: 1,optionally in combination with one or more additional amino acidsubstitutions, deletions or insertions relative to SEQ ID NO: 1.

Other GDF Trap proteins are contemplated, such as the following. A GDFTrap protein comprising an amino acid sequence that is at least 80%identical to the sequence of amino acids 29-109 of SEQ ID NO: 1 or 39,wherein the position corresponding to 64 of SEQ ID NO: 1 is an R or K,and wherein the GDF Trap protein inhibits signaling by activin,myostatin and/or GDF11 in a cell-based assay. The GDF Trap proteinabove, wherein at least one alteration with respect to the sequence ofSEQ ID NO: 1 or 39 is positioned outside of the ligand binding pocket.The GDF Trap protein above, wherein at least one alteration with respectto the sequence of SEQ ID NO: 1 or 39 is a conservative alterationpositioned within the ligand binding pocket. The GDF Trap protein above,wherein at least one alteration with respect to the sequence of SEQ IDNO: 1 or 39 is an alteration at one or more positions selected from thegroup consisting of K74, R40, Q53, K55, F82 and L79. The GDF Trapprotein above, wherein the protein comprises at least one N-X-S/Tsequence at a position other than an endogenous N-X-S/T sequence ofActRIIB, and at a position outside of the ligand binding pocket.

Other GDF Traps are contemplated, such as the following. A GDF Trapprotein comprising an amino acid sequence that is at least 80% identicalto the sequence of amino acids 29-109 of SEQ ID NO: 1 or 39, and whereinthe protein comprises at least one N-X-S/T sequence at a position otherthan an endogenous N-X-S/T sequence of ActRIIB, and at a positionoutside of the ligand binding pocket. The GDF Trap above, wherein theGDF Trap protein comprises an N at the position corresponding toposition 24 of SEQ ID NO: 1 or 39 and an S or T at the positioncorresponding to position 26 of SEQ ID NO: 1 or 39, and wherein the GDFTrap inhibits signaling by activin, myostatin and/or GDF11 in acell-based assay. The GDF Trap above, wherein the GDF Trap proteincomprises an R or K at the position corresponding to position 64 of SEQID NO: 1 or 39. The GDF Trap above, wherein the ActRIIB proteincomprises a D or E at the position corresponding to position 79 of SEQID NO: 1 or 39 and wherein the GDF Trap inhibits signaling by activin,myostatin and/or GDF11 in a cell-based assay. The GDF Trap above,wherein at least one alteration with respect to the sequence of SEQ IDNO: 1 or 39 is a conservative alteration positioned within the ligandbinding pocket. The GDF Trap above, wherein at least one alteration withrespect to the sequence of SEQ ID NO: 1 or 39 is an alteration at one ormore positions selected from the group consisting of K74, R40, Q53, K55,F82 and L79. The GDF Trap above, wherein the protein is a fusion proteinfurther comprising a heterologous portion. Any of the above GDF Trapfusion proteins may be produced as a homodimer. Any of the above GDFTrap fusion proteins may have a heterologous portion that comprises aconstant region from an IgG heavy chain, such as an Fc domain.

In certain aspects, the disclosure provides nucleic acids encoding a GDFTrap polypeptide. An isolated polynucleotide may comprise a codingsequence for a soluble GDF Trap polypeptide, such as described above.For example, an isolated nucleic acid may include a sequence coding fora GDF Trap comprising an extracellular domain (e.g., ligand-bindingdomain) of an ActRIIB polypeptide having one or more sequence variationsand a sequence that would code for part or all of the transmembranedomain and/or the cytoplasmic domain of an ActRIIB polypeptide, but fora stop codon positioned within the transmembrane domain or thecytoplasmic domain, or positioned between the extracellular domain andthe transmembrane domain or cytoplasmic domain. For example, an isolatedpolynucleotide coding for a GDF Trap may comprise a full-length ActRIIBpolynucleotide sequence such as SEQ ID NO: 4 having one or morevariations, or a partially truncated version, said isolatedpolynucleotide further comprising a transcription termination codon atleast six hundred nucleotides before the 3′-terminus or otherwisepositioned such that translation of the polynucleotide gives rise to anextracellular domain optionally fused to a truncated portion of afull-length ActRIIB Nucleic acids disclosed herein may be operablylinked to a promoter for expression, and the disclosure provides cellstransformed with such recombinant polynucleotides. Preferably the cellis a mammalian cell such as a CHO cell.

In certain aspects, the disclosure provides methods for making a GDFTrap polypeptide. Such a method may include expressing any of thenucleic acids (e.g., SEQ ID NO: 5, 25, 27, 30 or 31) disclosed herein ina suitable cell, such as a Chinese hamster ovary (CHO) cell. Such amethod may comprise: a) culturing a cell under conditions suitable forexpression of the GDF Trap polypeptide, wherein said cell is transformedwith a GDF Trap expression construct; and b) recovering the GDF Trappolypeptide so expressed. GDF Trap polypeptides may be recovered ascrude, partially purified or highly purified fractions using any of thewell known techniques for obtaining protein from cell cultures.

In certain aspects, a GDF Trap polypeptide disclosed herein may be usedin a method for promoting red blood cell production or increasing redblood cell levels in a subject. In certain embodiments, the disclosureprovides methods for treating a disorder associated with low red bloodcell counts or low hemoglobin levels (e.g., an anemia, myelodysplasticsyndrome, etc.), or to promote red blood cell production, in patients inneed thereof. A method may comprise administering to a subject in needthereof an effective amount of a GDF Trap polypeptide. In certainaspects, the disclosure provides uses of GDF Trap polypeptides formaking a medicament for the treatment of a disorder or condition asdescribed herein.

In certain aspects, the disclosure provides methods for administering aGDF Trap polypeptide to a patient. In part, the disclosure demonstratesthat GDF Trap polypeptides can be used to increase red blood cell andhemoglobin levels. GDF Trap polypeptides may also be used for treatingor preventing other therapeutic uses such as promoting muscle growth. Incertain instances, when administering a GDF Trap polypeptide forpromoting muscle growth, it may be desirable to monitor the effects onred blood cells during administration of the GDF Trap polypeptide, or todetermine or adjust the dosing of the GDF Trap polypeptide, in order toreduce undesired effects on red blood cells. For example, increases inred blood cell levels, hemoglobin levels, or hematocrit levels may causeincreases in blood pressure.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows an alignment of the extracellular domains of human ActRIIA(SEQ ID NO: 15) and human ActRIIB (SEQ ID NO: 2) with the residues thatare deduced herein, based on composite analysis of multiple ActRIIB andActRIIA crystal structures to directly contact ligand (the ligandbinding pocket) indicated with boxes.

FIG. 2 shows a multiple sequence alignment of various vertebrate ActRIIBproteins and human ActRIIA (SEQ ID NOs: 16-23).

FIG. 3 shows the full amino acid sequence for the GDF Trap ActRIIB(L79D20-134)-hFc (SEQ ID NO: 43), including the TPA leader sequence (doubleunderlined), ActRIIB extracellular domain (residues 20-134 in SEQ ID NO:1; underlined), and hFc domain. The aspartate substituted at position 79in the native sequence is double underlined and highlighted, as is theglycine revealed by sequencing to be the N-terminal residue in themature fusion protein.

FIGS. 4A and 4B shows a nucleotide sequence encoding ActRIIB(L79D20-134)-hFc. SEQ ID NO: 25 corresponds to the sense strand, and SEQ IDNO: 33 corresponds to the antisense strand. The TPA leader (nucleotides1-66) is double underlined, and the ActRIIB extracellular domain(nucleotides 76-420) is underlined.

FIG. 5 shows the full amino acid sequence for the truncated GDF TrapActRIIB(L79D 25-131)-hFc (SEQ ID NO: 26), including the TPA leader(double underlined), truncated ActRIIB extracellular domain (residues25-131 in SEQ ID NO: 1; underlined), and hFc domain. The aspartatesubstituted at position 79 in the native sequence is double underlinedand highlighted, as is the glutamate revealed by sequencing to be theN-terminal residue in the mature fusion protein.

FIGS. 6A and 6B shows a nucleotide sequence encoding ActRIIB(L79D25-131)-hFc. SEQ ID NO: 27 corresponds to the sense strand, and SEQ IDNO: 34 corresponds to the antisense strand. The TPA leader (nucleotides1-66) is double underlined, and the truncated ActRIIB extracellulardomain (nucleotides 76-396) is underlined. The amino acid sequence forthe ActRIIB extracellular domain (SEQ ID NO: 44) is also shown.

FIG. 7 shows the amino acid sequence for the truncated GDF TrapActRIIB(L79D 25-131)-hFc without a leader (SEQ ID NO: 28). The truncatedActRIIB extracellular domain (residues 25-131 in SEQ ID NO: 1) isunderlined. The aspartate substituted at position 79 in the nativesequence is double underlined and highlighted, as is the glutamaterevealed by sequencing to be the N-terminal residue in the mature fusionprotein.

FIG. 8 shows the amino acid sequence for the truncated GDF TrapActRIIB(L79D 25-131) without the leader, hFc domain, and linker (SEQ IDNO: 29). The aspartate substituted at position 79 in the native sequenceis underlined and highlighted, as is the glutamate revealed bysequencing to be the N-terminal residue in the mature fusion protein.

FIGS. 9A and 9B shows an alternative nucleotide sequence encodingActRIIB(L79D 25-131)-hFc. SEQ ID NO: 30 corresponds to the sense strand,and SEQ ID NO: 35 corresponds to the antisense strand. The TPA leader(nucleotides 1-66) is double underlined, the truncated ActRIIBextracellular domain (nucleotides 76-396) is underlined, andsubstitutions in the wildtype nucleotide sequence of the extracellulardomain are double underlined and highlighted (compare with SEQ ID NO:27, FIG. 6). The amino acid sequence for the ActRIIB extracellulardomain (SEQ ID NO: 44) is also shown.

FIG. 10 shows nucleotides 76-396 (SEQ ID NO: 31) of the alternativenucleotide sequence shown in FIG. 9 (SEQ ID NO: 30). The same nucleotidesubstitutions indicated in FIG. 9 are also underlined and highlightedhere. SEQ ID NO: 31 encodes only the truncated ActRIIB extracellulardomain (corresponding to residues 25-131 in SEQ ID NO: 1) with a L79Dsubstitution, e.g., ActRIIB(L79D 25-131).

FIG. 11 shows the effect of ActRIIB(L79D 25-131)-hFc on hemoglobinconcentration in a mouse model of chemotherapy-induced anemia. Data aremeans±SEM. **, P<0.01 vs. paclitaxel at the same time point. This GDFTrap offset the anemia induced by paclitaxel treatment.

FIG. 12 shows the effect of ActRIIB(L79D 25-131)-hFc on red blood cell(RBC) levels in a unilaterally nephrectomized (NEPHX) mouse model ofchronic kidney disease. Data are means±SEM. ***, P<0.001 vs. baseline.This GDF Trap reversed the nephrectomy-induced anemia observed incontrol mice.

FIG. 13 shows the effect of ActRIIB(L79D 25-131)-hFc on red blood cell(RBC), hemoglobin (HGB), and hematocrit (HCT) levels in a unilaterallynephrectomized (NEPHX) mouse model of chronic kidney disease. Data aremean changes from baseline over 4 weeks (±SEM). *, P<0.05; **, P<0.01;***, P<0.001 vs. NEPHX controls. This GDF Trap prevented thenephrectomy-associated decline in these erythrocytic parameters,increasing each by a magnitude similar to that in kidney-intact (sham)mice.

FIG. 14 shows the effect of ActRIIB(L79D 25-131)-hFc on red blood cell(RBC) levels in a rat model of anemia induced by acute blood loss. Bloodremoval occurred on Day-1, with dosing on Days 0 and 3. Data aremeans±SEM. **, P<0.01; ***, P<0.001 vs. vehicle at same time point. ThisGDF Trap improved the rate and extent of recovery fromblood-loss-induced anemia.

FIG. 15 shows the effect of treatment with ActRIIB(L79D 20-134)-hFc(gray) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change in redblood cell concentration from baseline in cynomolgus monkey.VEH=vehicle. Data are means±SEM. n=4-8 per group.

FIG. 16 shows the effect of treatment with ActRIIB(L79D 20-134)-hFc(gray) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change inhematocrit from baseline in cynomolgus monkey. VEH=vehicle. Data aremeans±SEM. n=4-8 per group.

FIG. 17 shows the effect of treatment with ActRIIB(L79D 20-134)-hFc(gray) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change inhemoglobin concentration from baseline in cynomolgus monkey.VEH=vehicle. Data are means±SEM. n=4-8 per group.

FIG. 18 shows the effect of treatment with ActRIIB(L79D 20-134)-hFc(gray) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change incirculating reticulocyte concentration from baseline in cynomolgusmonkey. VEH=vehicle. Data are means±SEM. n=4-8 per group.

FIG. 19 shows the effect of combined treatment with erythropoietin (EPO)and ActRIIB(L79D 25-131)-hFc for 72 hours on hematocrit in mice. Dataare means±SEM (n=4 per group), and means that are significantlydifferent from each other (p<0.05, unpaired t-test) are designated bydifferent letters. Combined treatment increased hematocrit by 23%compared to vehicle, a synergistic increase greater than the sum of theseparate effects of EPO and ActRIIB(L79D 25-131)-hFc.

FIG. 20 shows the effect of combined treatment with EPO and ActRIIB(L79D25-131)-hFc for 72 hours on hemoglobin concentrations in mice. Data aremeans±SEM (n=4 per group), and means that are significantly differentfrom each other (p<0.05) are designated by different letters. Combinedtreatment increased hemoglobin concentrations by 23% compared tovehicle, which was also a synergistic effect.

FIG. 21 shows the effect of combined treatment with EPO and ActRIIB(L79D25-131)-hFc for 72 hours on red blood cell concentrations in mice. Dataare means±SEM (n=4 per group), and means that are significantlydifferent from each other (p<0.05) are designated by different letters.Combined treatment increased red blood cell concentrations by 20%compared to vehicle, which was also a synergistic effect.

FIG. 22 shows the effect of combined treatment with EPO and ActRIIB(L79D25-131)-hFc for 72 hours on numbers of erythropoietic precursor cells inmouse spleen. Data are means±SEM (n=4 per group), and means that aresignificantly different from each other (p<0.01) are designated bydifferent letters. Whereas EPO alone increased the number of basophilicerythroblasts (BasoE) dramatically at the expense of late-stageprecursor maturation, combined treatment increased BasoE numbers to alesser but still significant extent while supporting undiminishedmaturation of late-stage precursors.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

EPO is a glycoprotein hormone involved in the growth and maturation oferythroid progenitor cells into erythrocytes. EPO is produced by theliver during fetal life and by the kidney in adults. Decreasedproduction of EPO, which commonly occurs in adults as a consequence ofrenal failure, leads to anemia. EPO has been produced by geneticengineering techniques based on expression and secretion of the proteinfrom a host cell transfected with the EPO gene. Administration of suchrecombinant EPO has been effective in the treatment of anemia. Forexample, Eschbach et al. (1987, N Engl J Med 316:73) describe the use ofEPO to correct anemia caused by chronic renal failure.

Effects of EPO are mediated through its binding to, and activation of, acell surface receptor belonging to the cytokine receptor superfamily anddesignated the EPO receptor. The human and murine EPO receptors havebeen cloned and expressed (D'Andrea et al., 1989, Cell 57:277; Jones etal., 1990, Blood 76:31; Winkelman et al., 1990, Blood 76:24; WO90/08822/U.S. Pat. No. 5,278,065). The human EPO receptor gene encodes a483 amino acid transmembrane protein comprising an extracellular domainof approximately 224 amino acids and exhibits approximately 82% aminoacid sequence identity with the murine EPO receptor (See U.S. Pat. No.6,319,499). The cloned, full-length EPO receptor expressed in mammaliancells (66-72 kDa) binds EPO with an affinity (K_(D)=100-300 nM) similarto that of the native receptor on erythroid progenitor cells. Thus, thisform is thought to contain the main EPO binding determinant and isreferred to as the EPO receptor. By analogy with other closely relatedcytokine receptors, the EPO receptor is thought to dimerize upon agonistbinding. Nevertheless, the detailed structure of the EPO receptor, whichmay be a multimeric complex, and its specific mechanism of activationare not completely understood (U.S. Pat. No. 6,319,499).

Activation of the EPO receptor results in several biological effects.These include increased proliferation of immature erythroblasts,increased differentiation of immature erythroblasts, and decreasedapoptosis in erythroid progenitor cells (Liboi et al., 1993, Proc NatlAcad Sci USA 90:11351-11355; Koury et al., 1990, Science 248:378-381).The EPO receptor signal transduction pathways mediating proliferationand differentiation appear to be distinct (Noguchi et al., 1988, MolCell Biol 8:2604; Patel et al., 1992, J Biol Chem 1992, 267:21300; Liboiet al., ibid). Some results suggest that an accessory protein may berequired for mediation of the differentiation signal (Chiba et al.,1993, Nature 362:646; Chiba et al., 1993, Proc Natl Acad Sci USA90:11593); however, there is controversy regarding the role of accessoryproteins in differentiation since a constituitively activated form ofthe receptor can stimulate both proliferation and differentiation (Pharret al., 1993, Proc Natl Acad Sci USA 90:938).

EPO receptor activators include small-moleculeerythropoiesis-stimulating agents (ESAs) as well as EPO-based compounds.An example of the former is a dimeric peptide-based agonist covalentlylinked to polyethylene glycol (proprietary name Hematide), which hasshown erythropoiesis-stimulating properties in healthy volunteers and inpatients with both chronic kidney disease and endogenous anti-EPOantibodies (Stead et al., 2006, Blood 108:1830-1834; Macdougall et al.,2009, N Engl J Med 361:1848-1855). Other examples includenonpeptide-based ESAs (Qureshi et al., 1999, Proc Natl Acad Sci USA96:12156-12161).

EPO receptor activators also include compounds that stimulateerythropoiesis indirectly, without contacting EPO receptor itself, byenhancing production of endogenous EPO. For example, hypoxia-inducibletranscription factors (HIFs) are endogenous stimulators of EPO geneexpression that are suppressed (destabilized) under normoxic conditionsby cellular regulatory mechanisms. Therefore, inhibitors of HIF prolylhydroxylase enzymes are being investigated for EPO-inducing activity invivo. Other indirect activators of EPO receptor include inhibitors ofGATA-2 transcription factor (Nakano et al., 2004, Blood 104:4300-4307),which tonically inhibits EPO gene expression, and inhibitors ofhemopoietic cell phosphatase (HCP or SHP-1), which functions as anegative regulator of EPO receptor signal transduction (Klingmuller etal., 1995, Cell 80:729-738).

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The family is dividedinto two general branches: the BMP/GDF and the TGF-beta/Activin/BMP10branches, whose members have diverse, often complementary effects. Bymanipulating the activity of a member of the TGF-beta family, it isoften possible to cause significant physiological changes in anorganism. For example, the Piedmontese and Belgian Blue cattle breedscarry a loss-of-function mutation in the GDF8 (also called myostatin)gene that causes a marked increase in muscle mass. Grobet et al., NatGenet. 1997, 17(1):71-4. Furthermore, in humans, inactive alleles ofGDF8 are associated with increased muscle mass and, reportedly,exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

TGF-β signals are mediated by heteromeric complexes of type I and typeII serine/threonine kinase receptors, which phosphorylate and activatedownstream Smad proteins upon ligand stimulation (Massagué, 2000, Nat.Rev. Mol. Cell Biol. 1:169-178). These type I and type II receptors aretransmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine specificity. Type Ireceptors are essential for signaling. Type II receptors are requiredfor binding ligands and for expression of Type I receptors. Type I andII activin receptors form a stable complex after ligand binding,resulting in phosphorylation of Type I receptors by Type II receptors.

Two related Type II receptors (ActRII), ActRIIA and ActRIIB, have beenidentified as the Type II receptors for activins (Mathews and Vale,1991, Cell 65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besidesactivins, ActRIIA and ActRIIB can biochemically interact with severalother TGF-β family proteins, including BMP7, Nodal, GDF8, and GDF11(Yamashita et al., 1995, J. Cell Biol. 130:217-226; Lee and McPherron,2001, Proc. Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol.Cell 7: 949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is theprimary type I receptor for activins, particularly for activin A, andALK-7 may serve as a receptor for activins as well, particularly foractivin B. In certain embodiments, the present invention relates toantagonizing a ligand of ActRIIB receptors (also referred to as anActRIIB ligand) with a subject GDF Trap polypeptide. Exemplary ligandsof ActRIIB receptors include some TGF-β family members, such as activin,Nodal, GDF8, GDF11, and BMP7.

Activins are dimeric polypeptide growth factors that belong to theTGF-beta superfamily. There are three principal activin forms (A, B, andAB) that are homo/heterodimers of two closely related β subunits(β_(A)β_(A), β_(B)β_(B), and β_(A)β_(B), respectively). The human genomealso encodes an activin C and an activin E, which are primarilyexpressed in the liver, and heterodimeric forms containing β_(C) orβ_(E) are also known. In the TGF-beta superfamily, activins are uniqueand multifunctional factors that can stimulate hormone production inovarian and placental cells, support neuronal cell survival, influencecell-cycle progress positively or negatively depending on cell type, andinduce mesodermal differentiation at least in amphibian embryos (DePaoloet al., 1991, Proc Soc Ep Biol Med. 198:500-512; Dyson et al., 1997,Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963).Moreover, erythroid differentiation factor (EDF) isolated from thestimulated human monocytic leukemic cells was found to be identical toactivin A (Murata et al., 1988, PNAS, 85:2434). It has been suggestedthat activin A promotes erythropoiesis in the bone marrow. In severaltissues, activin signaling is antagonized by its related heterodimer,inhibin. For example, during the release of follicle-stimulating hormone(FSH) from the pituitary, activin promotes FSH secretion and synthesis,while inhibin prevents FSH secretion and synthesis. Other proteins thatmay regulate activin bioactivity and/or bind to activin includefollistatin (FS), follistatin-related protein (FSRP) andα₂-macroglobulin.

Nodal proteins have functions in mesoderm and endoderm induction andformation, as well as subsequent organization of axial structures suchas heart and stomach in early embryogenesis. It has been demonstratedthat dorsal tissue in a developing vertebrate embryo contributespredominantly to the axial structures of the notochord and pre-chordalplate while it recruits surrounding cells to form non-axial embryonicstructures. Nodal appears to signal through both type I and type IIreceptors and intracellular effectors known as Smad proteins. Recentstudies support the idea that ActRIIA and ActRIIB serve as type IIreceptors for Nodal (Sakuma et al., Genes Cells. 2002, 7:401-12). It issuggested that Nodal ligands interact with their co-factors (e.g.,cripto) to activate activin type I and type II receptors, whichphosphorylate Smad2. Nodal proteins are implicated in many eventscritical to the early vertebrate embryo, including mesoderm formation,anterior patterning, and left-right axis specification. Experimentalevidence has demonstrated that Nodal signaling activates pAR3-Lux, aluciferase reporter previously shown to respond specifically to activinand TGF-beta. However, Nodal is unable to induce pTlx2-Lux, a reporterspecifically responsive to bone morphogenetic proteins. Recent resultsprovide direct biochemical evidence that Nodal signaling is mediated byboth activin-TGF-beta pathway Smads, Smad2 and Smad3. Further evidencehas shown that the extracellular cripto protein is required for Nodalsignaling, making it distinct from activin or TGF-beta signaling.

Growth and Differentiation Factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highlyexpressed in the developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of the skeletal muscle (McPherron et al., Nature, 1997,387:83-90). Similar increases in skeletal muscle mass are evident innaturally occurring mutations of GDF8 in cattle (Ashmore et al., 1974,Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci., 1994,38:752-757; McPherron and Lee, Proc. Natl. Acad. Sci. USA, 1997,94:12457-12461; and Kambadur et al., Genome Res., 1997, 7:910-915) and,strikingly, in humans (Schuelke et al., N Engl J Med 2004; 350:2682-8).Studies have also shown that muscle wasting associated withHIV-infection in humans is accompanied by increases in GDF8 proteinexpression (Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). Inaddition, GDF8 can modulate the production of muscle-specific enzymes(e.g., creatine kinase) and modulate myoblast cell proliferation (WO00/43781). The GDF8 propeptide can noncovalently bind to the mature GDF8domain dimer, inactivating its biological activity (Miyazono et al.(1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J. Biol.Chem., 263; 7646-7654; and Brown et al. (1990) Growth Factors, 3:35-43). Other proteins which bind to GDF8 or structurally relatedproteins and inhibit their biological activity include follistatin, andpotentially, follistatin-related proteins (Gamer et al. (1999) Dev.Biol., 208: 222-232).

Growth and Differentiation Factor-11 (GDF11), also known as BMP11, is asecreted protein (McPherron et al., 1999, Nat. Genet. 22: 260-264).GDF11 is expressed in the tail bud, limb bud, maxillary and mandibulararches, and dorsal root ganglia during mouse development (Nakashima etal., 1999, Mech. Dev. 80: 185-189). GDF11 plays a unique role inpatterning both mesodermal and neural tissues (Gamer et al., 1999, DevBiol., 208:222-32). GDF11 was shown to be a negative regulator ofchondrogenesis and myogenesis in developing chick limb (Gamer et al.,2001, Dev Biol. 229:407-20). The expression of GDF11 in muscle alsosuggests its role in regulating muscle growth in a similar way to GDF8.In addition, the expression of GDF11 in brain suggests that GDF11 mayalso possess activities that relate to the function of the nervoussystem. Interestingly, GDF11 was found to inhibit neurogenesis in theolfactory epithelium (Wu et al., 2003, Neuron. 37:197-207). Hence, GDF11may have in vitro and in vivo applications in the treatment of diseasessuch as muscle diseases and neurodegenerative diseases (e.g.,amyotrophic lateral sclerosis).

Bone morphogenetic protein (BMP7), also called osteogenic protein-1(OP-1), is well known to induce cartilage and bone formation. Inaddition, BMP7 regulates a wide array of physiological processes. Forexample, BMP7 may be the osteoinductive factor responsible for thephenomenon of epithelial osteogenesis. It is also found that BMP7 playsa role in calcium regulation and bone homeostasis. Like activin, BMP7binds to Type II receptors, ActRIIA and ActRIIB However, BMP7 andactivin recruit distinct Type I receptors into heteromeric receptorcomplexes. The major BMP7 Type I receptor observed was ALK2, whileactivin bound exclusively to ALK4 (ActRIIB) BMP7 and activin eliciteddistinct biological responses and activated different Smad pathways(Macias-Silva et al., 1998, J Biol Chem. 273:25628-36).

As demonstrated herein, a GDF Trap polypeptide, which is a variantActRIIB polypeptide (ActRIIB), is more effective at increasing red bloodcell levels in vivo as compared to a wild-type soluble ActRIIBpolypeptide and has beneficial effects in a variety of models foranemias. Additionally, it is shown that the use of a GDF Trappolypeptide in combination with an EPO receptor activator causes asubstantial increase in red blood cell formation. It should be notedthat hematopoiesis is a complex process, regulated by a variety offactors, including erythropoietin, G-CSF and iron homeostasis. The terms“increase red blood cell levels” and “promote red blood cell formation”refer to clinically observable metrics, such as hematocrit, red bloodcell counts and hemoglobin measurements, and are intended to be neutralas to the mechanism by which such changes occur.

In addition to stimulating red blood cell levels, GDF Trap polypeptidesare useful for a variety of therapeutic applications, including, forexample, promoting muscle growth (see PCT Publication Nos. WO2006/012627 and WO 2008/097541, which are hereby incorporated byreference in their entirety). In certain instances, when administering aGDF Trap polypeptide for the purpose of increasing muscle, it may bedesirable to reduce or minimize effects on red blood cells. Bymonitoring various hematologic parameters in patients being treatedwith, or who are candidates for treatment with, a GDF Trap polypeptide,appropriate dosing (including amounts and frequency of administration)may be determined based on an individual patient's needs, baselinehematologic parameters, and purpose for treatment. Furthermore,therapeutic progress and effects on one or more hematologic parametersover time may be useful in managing patients being dosed with a GDF Trappolypeptide by facilitating patient care, determining appropriatemaintenance dosing (both amounts and frequency), etc.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

2. GDF Trap Polypeptides

In certain aspects, the invention relates to GDF Trap polypeptides,e.g., soluble variant ActRIIB polypeptides, including, for example,fragments, functional variants, and modified forms of ActRIIBpolypeptides. In certain embodiments, the GDF Trap polypeptides have atleast one similar or same biological activity as a correspondingwild-type ActRIIB polypeptide. For example, a GDF Trap polypeptide ofthe invention may bind to and inhibit the function of an ActRIIB ligand(e.g., activin A, activin AB, activin B, Nodal, GDF8, GDF11 or BMP7).Optionally, a GDF Trap polypeptide increases red blood cell levels.Examples of GDF Trap polypeptides include human ActRIIB precursorpolypeptides (SEQ ID NO: 1 or 39) having one or more sequencevariations, and soluble human ActRIIB polypeptides (e.g., SEQ ID NOs: 2,3, 7, 11, 26, 28, 29, 32, 37, 38, 40 and 41) having one or more sequencevariations. A GDF Trap refers to an ActRIIB polypeptide having adecreased affinity for activin relative to other ActRIIB ligands,including for example GDF11 and/or myostatin.

As used herein, the term “ActRIIB” refers to a family of activinreceptor type IIb (ActRIIB) proteins from any species and variantsderived from such ActRIIB proteins by mutagenesis or other modification.Reference to ActRIIB herein is understood to be a reference to any oneof the currently identified forms. Members of the ActRIIB family aregenerally transmembrane proteins, composed of a ligand-bindingextracellular domain with a cysteine-rich region, a transmembranedomain, and a cytoplasmic domain with predicted serine/threonine kinaseactivity. Amino acid sequences of human ActRIIA soluble extracellulardomain (provided for comparison) and ActRIIB soluble extracellulardomain are illustrated in FIG. 1.

The term “ActRIIB polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIB family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. See, for example,WO 2006/012627. For example, ActRIIB polypeptides include polypeptidesderived from the sequence of any known ActRIIB having a sequence atleast about 80% identical to the sequence of an ActRIIB polypeptide, andoptionally at least 85%, 90%, 95%, 97%, 99% or greater identity. Forexample, an ActRIIB polypeptide may bind to and inhibit the function ofan ActRIIB protein and/or activin. An ActRIIB polypeptide which is a GDFTrap may be selected for activity in promoting red blood cell formationin vivo. Examples of ActRIIB polypeptides include human ActRIIBprecursor polypeptide (SEQ ID NO: 1 and 39) and soluble human ActRIIBpolypeptides (e.g., SEQ ID NO: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38, 40and 41). Numbering of amino acids for all ActRIIB-related polypeptidesdescribed herein is based on the numbering for SEQ ID NO:1, unlessspecifically designated otherwise.

The human ActRIIB precursor protein sequence is as follows:

(SEQ ID NO: 1)

ENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQHPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSLIRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI

The signal peptide is single underlined; the extracellular domain is inbold and the potential N-linked glycosylation sites are in boxes.

A form with an alanine at position 64 is also reported in theliterature, as follows:

(SEQ ID NO: 39)

ENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQHPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSLIRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI

The human ActRIIB soluble (extracellular), processed polypeptidesequence is as follows:

(SEQ ID NO: 2) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA GGPEVTYEPPPTAPT

The alternative form with an A64 is as follows:

(SEQ ID NO: 40) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA GGPEVTYEPPPTAPT

In some conditions, the protein may be produced with an “SGR . . . ”sequence at the N-terminus. The C-terminal “tail” of the extracellulardomain is underlined. The sequence with the “tail” deleted (a Δ15sequence) is as follows:

(SEQ ID NO: 3) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA

The alternative form with an A64 is as follows:

(SEQ ID NO: 41) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA

In some conditions, the protein may be produced with an “SGR . . . ”sequence at the N-terminus. The nucleic acid sequence encoding a humanActRIIB precursor protein is as follows: (nucleotides 5-1543 of Genbankentry NM_001106)(the sequence as shown provides an alanine at position64, and may be modified to provide an arginine instead)

(SEQ ID NO: 4) ATGACGGCGCCCTGGGTGGCCCTCGCCCTCCTCTGGGGATCGCTGTGGCCCGGCTCTGGGCGTGGGGAGGCTGAGACACGGGAGTGCATCTACTACAACGCCAACTGGGAGCTGGAGCGCACCAACCAGAGCGGCCTGGAGCGCTGCGAAGGCGAGCAGGACAAGCGGCTGCACTGCTACGCCTCCTGGGCCAACAGCTCTGGCACCATCGAGCTCGTGAAGAAGGGCTGCTGGCTAGATGACTTCAACTGCTACGATAGGCAGGAGTGTGTGGCCACTGAGGAGAACCCCCAGGTGTACTTCTGCTGCTGTGAAGGCAACTTCTGCAACGAGCGCTTCACTCATTTGCCAGAGGCTGGGGGCCCGGAAGTCACGTACGAGCCACCCCCGACAGCCCCCACCCTGCTCACGGTGCTGGCCTACTCACTGCTGCCCATCGGGGGCCTTTCCCTCATCGTCCTGCTGGCCTTTTGGATGTACCGGCATCGCAAGCCCCCCTACGGTCATGTGGACATCCATGAGGACCCTGGGCCTCCACCACCATCCCCTCTGGTGGGCCTGAAGCCACTGCAGCTGCTGGAGATCAAGGCTCGGGGGCGCTTTGGCTGTGTCTGGAAGGCCCAGCTCATGAATGACTTTGTAGCTGTCAAGATCTTCCCACTCCAGGACAAGCAGTCGTGGCAGAGTGAACGGGAGATCTTCAGCACACCTGGCATGAAGCACGAGAACCTGCTACAGTTCATTGCTGCCGAGAAGCGAGGCTCCAACCTCGAAGTAGAGCTGTGGCTCATCACGGCCTTCCATGACAAGGGCTCCCTCACGGATTACCTCAAGGGGAACATCATCACATGGAACGAACTGTGTCATGTAGCAGAGACGATGTCACGAGGCCTCTCATACCTGCATGAGGATGTGCCCTGGTGCCGTGGCGAGGGCCACAAGCCGTCTATTGCCCACAGGGACTTTAAAAGTAAGAATGTATTGCTGAAGAGCGACCTCACAGCCGTGCTGGCTGACTTTGGCTTGGCTGTTCGATTTGAGCCAGGGAAACCTCCAGGGGACACCCACGGACAGGTAGGCACGAGACGGTACATGGCTCCTGAGGTGCTCGAGGGAGCCATCAACTTCCAGAGAGATGCCTTCCTGCGCATTGACATGTATGCCATGGGGTTGGTGCTGTGGGAGCTTGTGTCTCGCTGCAAGGCTGCAGACGGACCCGTGGATGAGTACATGCTGCCCTTTGAGGAAGAGATTGGCCAGCACCCTTCGTTGGAGGAGCTGCAGGAGGTGGTGGTGCACAAGAAGATGAGGCCCACCATTAAAGATCACTGGTTGAAACACCCGGGCCTGGCCCAGCTTTGTGTGACCATCGAGGAGTGCTGGGACCATGATGCAGAGGCTCGCTTGTCCGCGGGCTGTGTGGAGGAGCGGGTGTCCCTGATTCGGAGGTCGGTCAACGGCACTACCTCGGACTGTCTCGTTTCCCTGGTGACCTCTGTCACCAATGTGGACCTGCCCCCTAAAGAGTCAAGCATCTAAThe nucleic acid sequence encoding a human ActRIIB soluble(extracellular) polypeptide is as follows (the sequence as shownprovides an alanine at position 64, and may be modified to provide anarginine instead):

(SEQ ID NO: 5) GGGCGTGGGGAGGCTGAGACACGGGAGTGCATCTACTACAACGCCAACTGGGAGCTGGAGCGCACCAACCAGAGCGGCCTGGAGCGCTGCGAAGGCGAGCAGGACAAGCGGCTGCACTGCTACGCCTCCTGGGCCAACAGCTCTGGCACCATCGAGCTCGTGAAGAAGGGCTGCTGGCTAGATGACTTCAACTGCTACGATAGGCAGGAGTGTGTGGCCACTGAGGAGAACCCCCAGGTGTACTTCTGCTGCTGTGAAGGCAACTTCTGCAACGAGCGCTTCACTCATTTGCCAGAGGCTGGGGGCCCGGAAGTCACGTACGAGCCACCCCCGACAGCCCCCACC

In a specific embodiment, the invention relates to GDF Trap polypeptideswhich are variant forms of soluble ActRIIB polypeptides. As describedherein, the term “soluble ActRIIB polypeptide” generally refers topolypeptides comprising an extracellular domain of an ActRIIB protein.The term “soluble ActRIIB polypeptide,” as used herein, includes anynaturally occurring extracellular domain of an ActRIIB protein as wellas any variants thereof (including mutants, fragments and peptidomimeticforms) that retain a useful activity. For example, the extracellulardomain of an ActRIIB protein binds to a ligand and is generally soluble.Examples of soluble ActRIIB polypeptides include ActRIIB solublepolypeptides (e.g., SEQ ID NOs: 2, 3, 7, 11, 26, 28, 29, 32, 37, 38, 40and 41). Other examples of soluble ActRIIB polypeptides comprise asignal sequence in addition to the extracellular domain of an ActRIIBprotein, see Example 1. The signal sequence can be a native signalsequence of an ActRIIB, or a signal sequence from another protein, suchas a tissue plasminogen activator (TPA) signal sequence or a honey beemelittin (HBM) signal sequence.

The disclosure identifies functionally active portions and variants ofActRIIB Applicants have ascertained that an Fc fusion protein having thesequence disclosed by Hilden et al. (Blood. 1994 Apr. 15;83(8):2163-70), which has an Alanine at the position corresponding toamino acid 64 of SEQ ID NO: 1 (A64), has a relatively low affinity foractivin and GDF-11. By contrast, the same Fc fusion protein with anArginine at position 64 (R64) has an affinity for activin and GDF-11 inthe low nanomolar to high picomolar range. Therefore, a sequence with anR64 is used as the wild-type reference sequence for human ActRIIB inthis disclosure.

Attisano et al. (Cell. 1992 Jan. 10; 68(1):97-108) showed that adeletion of the proline knot at the C-terminus of the extracellulardomain of ActRIIB reduced the affinity of the receptor for activin. AnActRIIB-Fc fusion protein containing amino acids 20-119 of SEQ ID NO: 1,“ActRIIB(20-119)-Fc”, has reduced binding to GDF-11 and activin relativeto an ActRIIB(20-134)-Fc, which includes the proline knot region and thecomplete juxtamembrane domain. However, an ActRIIB(20-129)-Fc proteinretains similar but somewhat reduced activity relative to the wild type,even though the proline knot region is disrupted. Thus, ActRIIBextracellular domains that stop at amino acid 134, 133, 132, 131, 130and 129 are all expected to be active, but constructs stopping at 134 or133 may be most active. Similarly, mutations at any of residues 129-134are not expected to alter ligand binding affinity by large margins. Insupport of this, mutations of P129 and P130 do not substantiallydecrease ligand binding. Therefore, a GDF Trap polypeptide which is anActRIIB-Fc fusion protein may end as early as amino acid 109 (the finalcysteine), however, forms ending at or between 109 and 119 are expectedto have reduced ligand binding. Amino acid 119 is poorly conserved andso is readily altered or truncated. Forms ending at 128 or later retainligand binding activity. Forms ending at or between 119 and 127 willhave an intermediate binding ability. Any of these forms may bedesirable to use, depending on the clinical or experimental setting.

At the N-terminus of ActRIIB, it is expected that a protein beginning atamino acid 29 or before will retain ligand binding activity. Amino acid29 represents the initial cysteine. An alanine to asparagine mutation atposition 24 introduces an N-linked glycosylation sequence withoutsubstantially affecting ligand binding. This confirms that mutations inthe region between the signal cleavage peptide and the cysteinecross-linked region, corresponding to amino acids 20-29 are welltolerated. In particular, constructs beginning at position 20, 21, 22,23 and 24 will retain activity, and constructs beginning at positions25, 26, 27, 28 and 29 are also expected to retain activity. Data shownin the Examples demonstrates that, surprisingly, a construct beginningat 22, 23, 24 or 25 will have the most activity.

Taken together, an active portion of ActRIIB comprises amino acids29-109 of SEQ ID NO: 1, and GDF Trap constructs may, for example,comprise a portion of ActRIIB beginning at a residue corresponding toamino acids 20-29 of SEQ ID NO: 1 or 39 and ending at a positioncorresponding to amino acids 109-134 of SEQ ID NO: 1 or 39. Otherexamples include constructs that begin at a position from 20-29 or 21-29and end at a position from 119-134, 119-133, 129-134, or 129-133 of SEQID NO: 1 or 39. Other examples include constructs that begin at aposition from 20-24 (or 21-24, or 22-25) and end at a position from109-134 (or 109-133), 119-134 (or 119-133) or 129-134 (or 129-133) ofSEQ ID NO: 1 or 39. Variants within these ranges are also contemplated,particularly those having at least 80%, 85%, 90%, 95% or 99% identity tothe corresponding portion of SEQ ID NO: 1 or 39. In certain embodiments,the GDF Trap polypeptide comprises, consists essentially of, or consistsof, a polypeptide having an amino acid sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acidresidues 25-131 of SEQ ID NO: 1 or 39. In certain embodiments, the GDFTrap polypeptide comprises, consists essentially of, or consists of, apolypeptide having an amino acid sequence that is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 7, 26, 28,29, 32, 37 or 38. In preferred embodiments, the GDF Trap polypeptideconsists of, or consists essentially of, the amino acid sequence of SEQID NO: 7, 26, 28, 29, 32, 37 or 38.

The disclosure includes the results of an analysis of composite ActRIIBstructures, shown in FIG. 1, demonstrating that the ligand bindingpocket is defined by residues Y31, N33, N35, L38 through T41, E47, E50,Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83, Y85, R87,A92, and E94 through F101. At these positions, it is expected thatconservative mutations will be tolerated, although a K74A mutation iswell-tolerated, as are R40A, K55A, F82A and mutations at position L79.R40 is a K in Xenopus, indicating that basic amino acids at thisposition will be tolerated. Q53 is R in bovine ActRIIB and K in XenopusActRIIB, and therefore amino acids including R, K, Q, N and H will betolerated at this position. Thus, a general formula for a GDF Trapprotein is one that comprises amino acids 29-109 of SEQ ID NO: 1 or 39,but optionally beginning at a position ranging from 20-24 or 22-25 andending at a position ranging from 129-134, and comprising no more than1, 2, 5, 10 or 15 conservative amino acid changes in the ligand bindingpocket, and zero, one or more non-conservative alterations at positions40, 53, 55, 74, 79 and/or 82 in the ligand binding pocket. Such aprotein may retain greater than 80%, 90%, 95% or 99% sequence identityto the sequence of amino acids 29-109 of SEQ ID NO: 1 or 39. Sitesoutside the binding pocket, at which variability may be particularlywell tolerated, include the amino and carboxy termini of theextracellular domain (as noted above), and positions 42-46 and 65-73. Anasparagine to alanine alteration at position 65 (N65A) actually improvesligand binding in the A64 background, and is thus expected to have nodetrimental effect on ligand binding in the R64 background. This changeprobably eliminates glycosylation at N65 in the A64 background, thusdemonstrating that a significant change in this region is likely to betolerated. While an R64A change is poorly tolerated, R64K iswell-tolerated, and thus another basic residue, such as H may betolerated at position 64.

ActRIIB is well-conserved across nearly all vertebrates, with largestretches of the extracellular domain conserved completely. Many of theligands that bind to ActRIIB are also highly conserved. Accordingly,comparisons of ActRIIB sequences from various vertebrate organismsprovide insights into residues that may be altered. Therefore, anactive, human ActRIIB variant polypeptide useful as a GDF Trap mayinclude one or more amino acids at corresponding positions from thesequence of another vertebrate ActRIIB, or may include a residue that issimilar to that in the human or other vertebrate sequence. The followingexamples illustrate this approach to defining an active ActRIIB variant.L46 is a valine in Xenopus ActRIIB, and so this position may be altered,and optionally may be altered to another hydrophobic residue, such as V,I or F, or a non-polar residue such as A. E52 is a K in Xenopus,indicating that this site may be tolerant of a wide variety of changes,including polar residues, such as E, D, K, R, H, S, T, P, G, Y andprobably A. T93 is a K in Xenopus, indicating that a wide structuralvariation is tolerated at this position, with polar residues favored,such as S, K, R, E, D, H, G, P, G and Y. F108 is a Y in Xenopus, andtherefore Y or other hydrophobic group, such as I, V or L should betolerated. E111 is K in Xenopus, indicating that charged residues willbe tolerated at this position, including D, R, K and H, as well as Q andN. R112 is K in Xenopus, indicating that basic residues are tolerated atthis position, including R and H. A at position 119 is relatively poorlyconserved, and appears as P in rodents and V in Xenopus, thusessentially any amino acid should be tolerated at this position.

The disclosure demonstrates that the addition of a further N-linkedglycosylation site (N-X-S/T) increases the serum half-life of anActRIIB-Fc fusion protein, relative to the ActRIIB(R64)-Fc form. Byintroducing an asparagine at position 24 (A24N construct), an NXTsequence is created that confers a longer half-life. Other NX(T/S)sequences are found at 42-44 (NQS) and 65-67 (NSS), although the lattermay not be efficiently glycosylated with the R at position 64. N-X-S/Tsequences may be generally introduced at positions outside the ligandbinding pocket defined in FIG. 1. Particularly suitable sites for theintroduction of non-endogenous N-X-S/T sequences include amino acids20-29, 20-24, 22-25, 109-134, 120-134 or 129-134. N-X-S/T sequences mayalso be introduced into the linker between the ActRIIB sequence and theFc or other fusion component. Such a site may be introduced with minimaleffort by introducing an N in the correct position with respect to apre-existing S or T, or by introducing an S or T at a positioncorresponding to a pre-existing N. Thus, desirable alterations thatwould create an N-linked glycosylation site are: A24N, R64N, S67N(possibly combined with an N65A alteration), E106N, R112N, G120N, E123N,P129N, A132N, R112S and R112T. Any S that is predicted to beglycosylated may be altered to a T without creating an immunogenic site,because of the protection afforded by the glycosylation. Likewise, any Tthat is predicted to be glycosylated may be altered to an S. Thus thealterations S67T and S44T are contemplated. Likewise, in an A24Nvariant, an S26T alteration may be used. Accordingly, a GDF Trap may bean ActRIM variant having one or more additional, non-endogenous N-linkedglycosylation consensus sequences.

Position L79 of ActRIM may be altered to confer alteredactivin—myostatin (GDF-11) binding properties. L79A or L79P reducesGDF-11 binding to a greater extent than activin binding. L79E or L79Dretains GDF-11 binding. Remarkably, the L79E and L79D variants havegreatly reduced activin binding. In vivo experiments indicate that thesenon-activin receptors retain significant ability to increase red bloodcells but show decreased effects on other tissues. These datademonstrate the desirability and feasibility for obtaining polypeptideswith reduced effects on activin. In exemplary embodiments, the methodsdescribed herein utilize a GDF Trap polypeptide which is a variantActRIIB polypeptide comprising an acidic amino acid (e.g., D or E) atthe position corresponding to position 79 of SEQ ID NO: 1 or 39,optionally in combination with one or more additional amino acidsubstitutions, additions, or deletions.

The variations described may be combined in various ways. Additionally,the results of the mutagenesis program described herein indicate thatthere are amino acid positions in ActRIIB that are often beneficial toconserve. These include position 64 (basic amino acid), position 80(acidic or hydrophobic amino acid), position 78 (hydrophobic, andparticularly tryptophan), position 37 (acidic, and particularly asparticor glutamic acid), position 56 (basic amino acid), position 60(hydrophobic amino acid, particularly phenylalanine or tyrosine). Thus,in each of the variants disclosed herein, the disclosure provides aframework of amino acids that may be conserved. Other positions that maybe desirable to conserve are as follows: position 52 (acidic aminoacid), position 55 (basic amino acid), position 81 (acidic), 98 (polaror charged, particularly E, D, R or K).

In certain embodiments, isolated fragments of ActRIIB polypeptides canbe obtained by screening polypeptides recombinantly produced from thecorresponding fragment of the nucleic acid encoding an ActRIIBpolypeptide (e.g., SEQ ID NOs: 4 and 5). In addition, fragments can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Thefragments can be produced (recombinantly or by chemical synthesis) andtested to identify those peptidyl fragments that can function, forexample, as antagonists (inhibitors) or agonists (activators) of anActRIIB protein or an ActRIIB ligand.

In certain embodiments, GDF Trap polypeptide is a variant ActRIIBpolypeptide having an amino acid sequence that is at least 75% identicalto an amino acid sequence selected from SEQ ID NOs: 2, 3, 7, 11, 26, 28,29, 32, 37, 38, 40 or 41. In certain cases, the GDF Trap has an aminoacid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from SEQ ID NOs: 2, 3, 7,11, 26, 28, 29, 32, 37, 38, 40 or 41. In certain emobdiments, the GDFTrap comprises, consists essentially of, or consists of, an amino acidsequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical toan amino acid sequence selected from SEQ ID NOs: 2, 3, 7, 11, 26, 28,29, 32, 37, 38, 40 or 41, wherein the position corresponding to L79 ofSEQ ID NO: 1 is an acidic amino acid (e.g., a D or E amino acidresidue).

In certain embodiments, the present invention contemplates makingfunctional variants by modifying the structure of a GDF Trap polypeptidefor such purposes as enhancing therapeutic efficacy, or stability (e.g.,ex vivo shelf life and resistance to proteolytic degradation in vivo).GDF Trap polypeptides can also be produced by amino acid substitution,deletion, or addition. For instance, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid(e.g., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Whether a change in the amino acidsequence of a GDF Trap polypeptide results in a functional variant canbe readily determined by assessing the ability of the GDF Trappolypeptide to produce a response in cells relative to the unmodifiedGDF Trap polypeptide or a wild-type ActRIIB polypeptide, or to bind toone or more ligands, such as activin, GDF-11 or myostatin as compared tothe unmodified GDF Trap polypeptide or a wild-type ActRIIB polypeptide.

In certain specific embodiments, the present invention contemplatesmaking mutations in the extracellular domain (also referred to asligand-binding domain) of an ActRIIB polypeptide such that the ActRIIBpolypeptide has altered ligand-binding activities (e.g., bindingaffinity or binding specificity). In certain cases, such GDF Trappolypeptides have altered (elevated or reduced) binding affinity for aspecific ligand. In other cases, the GDF Trap polypeptides have alteredbinding specificity for ActRIIB ligands.

For example, the disclosure provides GDF Trap polypeptides thatpreferentially bind to GDF8/GDF11 relative to activins. The disclosurefurther establishes the desirability of such polypeptides for reducingoff-target effects, although such selective variants may be lessdesirable for the treatment of severe diseases where very large gains inred blood cell levels may be needed for therapeutic effect and wheresome level of off-target effect is acceptable. For example, amino acidresidues of the ActRIIB protein, such as E39, K55, Y60, K74, W78, D80,and F101, are in the ligand-binding pocket and mediate binding to itsligands such as activin and GDF8. Thus, the present invention provides aGDF Trap comprising an altered ligand-binding domain (e.g., GDF8-bindingdomain) of an ActRIIB receptor, which comprises one or more mutations atthose amino acid residues. Optionally, the altered ligand-binding domaincan have increased selectivity for a ligand such as GDF8 relative to awild-type ligand-binding domain of an ActRIIB receptor. To illustrate,these mutations increase the selectivity of the altered ligand-bindingdomain for GDF8 over activin. Optionally, the altered ligand-bindingdomain has a ratio of K_(d) for activin binding to K_(d) for GDF8binding that is at least 2, 5, 10, or even 100 fold greater relative tothe ratio for the wild-type ligand-binding domain. Optionally, thealtered ligand-binding domain has a ratio of IC₅₀ for inhibiting activinto IC₅₀ for inhibiting GDF8 that is at least 2, 5, 10, or even 100 foldgreater relative to the wild-type ligand-binding domain. Optionally, thealtered ligand-binding domain inhibits GDF8 with an IC₅₀ at least 2, 5,10, or even 100 times less than the IC₅₀ for inhibiting activin.

As a specific example, the positively-charged amino acid residue Asp(D80) of the ligand-binding domain of ActRIIB can be mutated to adifferent amino acid residue to produce a GDF Trap polypeptide thatpreferentially binds to GDF8, but not activin. Preferably, the D80residue is changed to an amino acid residue selected from the groupconsisting of: an uncharged amino acid residue, a negative amino acidresidue, and a hydrophobic amino acid residue. As a further specificexample, the hydrophobic residue, L79, can be altered to the acidicamino acids aspartic acid or glutamic acid to greatly reduce activinbinding while retaining GDF11 binding. As will be recognized by one ofskill in the art, most of the described mutations, variants ormodifications may be made at the nucleic acid level or, in some cases,by post translational modification or chemical synthesis. Suchtechniques are well known in the art.

In certain embodiments, the present invention contemplates GDF Trappolypeptides having specific mutations in ActRIIB so as to alter theglycosylation of the ActRIIB polypeptide. Exemplary glycosylation sitesin GDF Trap polypeptides are illustrated in FIG. 1 (e.g., the underlinedNX(S/T) sites). Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, such as O-linked or N-linkedglycosylation sites. Asparagine-linked glycosylation recognition sitesgenerally comprise a tripeptide sequence, asparagine-X-threonine (where“X” is any amino acid) which is specifically recognized by appropriatecellular glycosylation enzymes. The alteration may also be made by theaddition of, or substitution by, one or more serine or threonineresidues to the sequence of the wild-type ActRIIB polypeptide (forO-linked glycosylation sites). A variety of amino acid substitutions ordeletions at one or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. Another means of increasing the number of carbohydratemoieties on a GDF Trap polypeptide is by chemical or enzymatic couplingof glycosides to the GDF Trap polypeptide. Depending on the couplingmode used, the sugar(s) may be attached to (a) arginine and histidine;(b) free carboxyl groups; (c) free sulfhydryl groups such as those ofcysteine; (d) free hydroxyl groups such as those of serine, threonine,or hydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Thesemethods are described in WO 87/05330 and in Aplin and Wriston (1981) CRCCrit. Rev. Biochem., pp. 259-306, incorporated by reference herein.Removal of one or more carbohydrate moieties present on a GDF Trappolypeptide may be accomplished chemically and/or enzymatically.Chemical deglycosylation may involve, for example, exposure of the GDFTrap polypeptide to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the amino acid sequence intact.Chemical deglycosylation is further described by Hakimuddin et al.(1987) Arch. Biochem. Biophys. 259:52 and by Edge et al. (1981) Anal.Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on GDFTrap polypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol.138:350. The sequence of a GDF Trap polypeptide may be adjusted, asappropriate, depending on the type of expression system used, asmammalian, yeast, insect and plant cells may all introduce differingglycosylation patterns that can be affected by the amino acid sequenceof the peptide. In general, GDF Trap polypeptides for use in humans willbe expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines are expected to be useful as well.

This disclosure further contemplates a method of generating variants,particularly sets of combinatorial variants of a GDF Trap polypeptide,including, optionally, truncation variants; pools of combinatorialmutants are especially useful for identifying GDF Trap sequences. Thepurpose of screening such combinatorial libraries may be to generate,for example, GDF Trap polypeptide variants which have alteredproperties, such as altered pharmacokinetics, or altered ligand binding.A variety of screening assays are provided below, and such assays may beused to evaluate variants. For example, a GDF Trap polypeptide variantmay be screened for the ability to bind to an ActRIIB polypeptide, toprevent binding of an ActRIIB ligand to an ActRIIB polypeptide or tointerfere with signaling caused by an ActRIIB ligand.

The activity of a GDF Trap polypeptide or its variants may also betested in a cell-based or in vivo assay. For example, the effect of aGDF Trap polypeptide variant on the expression of genes involved inhematopoiesis may be assessed. This may, as needed, be performed in thepresence of one or more recombinant ActRIIB ligand proteins (e.g.,activin), and cells may be transfected so as to produce a GDF Trappolypeptide and/or variants thereof, and optionally, an ActRIIB ligand.Likewise, a GDF Trap polypeptide may be administered to a mouse or otheranimal, and one or more blood measurements, such as an RBC count,hemoglobin levels, hematocrit levels, iron stores, or reticulocyte countmay be assessed using art recognized methods.

Combinatorially-derived variants can be generated which have a selectivepotency relative to a reference GDF Trap polypeptide. Such variantproteins, when expressed from recombinant DNA constructs, can be used ingene therapy protocols. Likewise, mutagenesis can give rise to variantswhich have intracellular half-lives dramatically different than thecorresponding unmodified GDF Trap polypeptide. For example, the alteredprotein can be rendered either more stable or less stable to proteolyticdegradation or other processes which result in destruction of, orotherwise inactivation of an unmodified GDF Trap polypeptide. Suchvariants, and the genes which encode them, can be utilized to alter GDFTrap polypeptide levels by modulating the half-life of the GDF Trappolypeptides. For instance, a short half-life can give rise to moretransient biological effects and, when part of an inducible expressionsystem, can allow tighter control of recombinant GDF Trap polypeptidelevels within the cell. In an Fc fusion protein, mutations may be madein the linker (if any) and/or the Fc portion to alter the half-life ofthe protein.

In certain embodiments, the GDF Trap polypeptides of the invention mayfurther comprise post-translational modifications in addition to anythat are naturally present in the ActRIIB polypeptides. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, GDF Trap polypeptides may contain non-amino acidelements, such as polyethylene glycols, lipids, poly- ormono-saccharide, and phosphates. Effects of such non-amino acid elementson the functionality of a GDF Trap polypeptide may be tested asdescribed herein for other GDF Trap polypeptide variants. When a GDFTrap polypeptide is produced in cells by cleaving a nascent form of theGDF Trap polypeptide, post-translational processing may also beimportant for correct folding and/or function of the protein. Differentcells (such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) havespecific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the GDF Trap polypeptides.

In certain aspects, GDF Trap polypeptides include fusion proteins havingat least a portion of an ActRIIB polypeptide and one or more fusiondomains. Well known examples of such fusion domains include, but are notlimited to, polyhistidine, Glu-Glu, glutathione S transferase (GST),thioredoxin, protein A, protein G, an immunoglobulin heavy chainconstant region (e.g., an Fc), maltose binding protein (MBP), or humanserum albumin. A fusion domain may be selected so as to confer a desiredproperty. For example, some fusion domains are particularly useful forisolation of the fusion proteins by affinity chromatography. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Many of such matrices are availablein “kit” form, such as the Pharmacia GST purification system and theQIAexpress™ system (Qiagen) useful with (HIS₆) (SEQ ID NO: 24) fusionpartners. As another example, a fusion domain may be selected so as tofacilitate detection of the GDF Trap polypeptides. Examples of suchdetection domains include the various fluorescent proteins (e.g., GFP)as well as “epitope tags,” which are usually short peptide sequences forwhich a specific antibody is available. Well known epitope tags forwhich specific monoclonal antibodies are readily available include FLAG,influenza virus haemagglutinin (HA), and c-myc tags. In some cases, thefusion domains have a protease cleavage site, such as for Factor Xa orThrombin, which allows the relevant protease to partially digest thefusion proteins and thereby liberate the recombinant proteins therefrom.The liberated proteins can then be isolated from the fusion domain bysubsequent chromatographic separation. In certain preferred embodiments,a GDF Trap polypeptide is fused with a domain that stabilizes the GDFTrap polypeptide in vivo (a “stabilizer” domain). By “stabilizing” ismeant anything that increases serum half life, regardless of whetherthis is because of decreased destruction, decreased clearance by thekidney, or other pharmacokinetic effect. Fusions with the Fc portion ofan immunoglobulin are known to confer desirable pharmacokineticproperties on a wide range of proteins. Likewise, fusions to human serumalbumin can confer desirable properties. Other types of fusion domainsthat may be selected include multimerizing (e.g., dimerizing,tetramerizing) domains and functional domains (that confer an additionalbiological function, such as further increasing red blood cell levels).

As a specific example, the present invention provides GDF Trap that isan ActRIIB-Fc fusion protein which comprises an extracellular (e.g.,ligand-binding) domain of ActRIIB polypeptide fused to an Fc domain. Thesequence of an exemplary Fc domain is shown below (SEQ ID NO: 6).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

Optionally, the Fc domain has one or more mutations at residues such asAsp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asp-265 mutation) hasreduced ability of binding to the Fcγ receptor relative to a wildtype Fcdomain. In other cases, the mutant Fc domain having one or more of thesemutations (e.g., Asn-434 mutation) has increased ability of binding tothe MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fcdomain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, a GDF Trap polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to a GDF Trap polypeptide. The GDF Trappolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

In certain embodiments, a GDF Trap fusion protein comprises an aminoacid sequence as set forth in the formula A-B-C. The B portion is an N-and C-terminally truncated ActRIIB polypeptide consisting of the aminoacid sequence corresponding to amino acids 26-132 of SEQ ID NO: 26. TheA and C portions may be independently zero, one or more than one aminoacids, and both the A and C portions when present are heterologous to B.The A and/or C portions may be attached to the B portion via a linkersequence. Exemplary linkers are include short polypeptide linkers suchas 2-10, 2-5, 2-4, 2-3 Glycine residues, such as, for example, aGly-Gly-Gly linker. Other suitable linkers are described herein above.In certain embodiments, a GDF Trap fusion protein comprises an aminoacid sequence as set forth in the formula A-B-C, wherein A is a leadersequence, B consists of amino acids 26-132 of SEQ ID NO: 26, and C is apolypeptide portion that enhances one or more of in vivo stability, invivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, and/or purification. Incertain embodiments, a GDF Trap fusion protein comprises an amino acidsequence as set forth in the formula A-B-C, wherein A is a TPA leadersequence, B consists of amino acids 26-132 of SEQ ID NO: 26, and C is animmunoglobulin Fc domain. A preferred GDF Trap fusion protein comprisesthe amino acid sequence set forth in SEQ ID NO: 26.

In certain embodiments, the GDF Trap polypeptides of the presentinvention contain one or more modifications that are capable ofstabilizing the GDF Trap polypeptides. For example, such modificationsenhance the in vitro half life of the GDF Trap polypeptides, enhancecirculatory half life of the GDF Trap polypeptides or reducingproteolytic degradation of the GDF Trap polypeptides. Such stabilizingmodifications include, but are not limited to, fusion proteins(including, for example, fusion proteins comprising an GDF Trappolypeptide and a stabilizer domain), modifications of a glycosylationsite (including, for example, addition of a glycosylation site to a GDFTrap polypeptide), and modifications of carbohydrate moiety (including,for example, removal of carbohydrate moieties from a GDF Trappolypeptide). In the case of fusion proteins, a GDF Trap polypeptide isfused to a stabilizer domain such as an IgG molecule (e.g., an Fcdomain). As used herein, the term “stabilizer domain” not only refers toa fusion domain (e.g., Fc) as in the case of fusion proteins, but alsoincludes nonproteinaceous modifications such as a carbohydrate moiety,or nonproteinaceous polymer, such as polyethylene glycol.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the GDF Trap polypeptides, which are isolatedfrom, or otherwise substantially free of, other proteins.

In certain embodiments, GDF Trap polypeptides (unmodified or modified)of the invention can be produced by a variety of art-known techniques.For example, such GDF Trap polypeptides can be synthesized usingstandard protein chemistry techniques such as those described inBodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin(1993) and Grant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H.Freeman and Company, New York (1992). In addition, automated peptidesynthesizers are commercially available (e.g., Advanced ChemTech Model396; Milligen/Biosearch 9600). Alternatively, the GDF Trap polypeptides,fragments or variants thereof may be recombinantly produced usingvarious expression systems (e.g., E. coli, Chinese Hamster Ovary (CHO)cells, COS cells, baculovirus) as is well known in the art. In a furtherembodiment, the modified or unmodified GDF Trap polypeptides may beproduced by digestion of recombinantly produced full-length GDF Trappolypeptides by using, for example, a protease, e.g., trypsin,thermolysin, chymotrypsin, pepsin, or paired basic amino acid convertingenzyme (PACE). Computer analysis (using a commercially availablesoftware, e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.)can be used to identify proteolytic cleavage sites. Alternatively, suchGDF Trap polypeptides may be produced from recombinantly producedfull-length GDF Trap polypeptides such as standard techniques known inthe art, such as by chemical cleavage (e.g., cyanogen bromide,hydroxylamine).

3. Nucleic Acids Encoding GDF Trap Polypeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the GDF Trap polypeptides disclosedherein. SEQ ID NO: 4 encodes a naturally occurring ActRIIB precursorpolypeptide, while SEQ ID NO: 5 encodes a soluble ActRIIB polypeptide,and SEQ ID NOs: 25, 27, 30 and 31 encode soluble GDF Traps. The subjectnucleic acids may be single-stranded or double stranded. Such nucleicacids may be DNA or RNA molecules. These nucleic acids may be used, forexample, in methods for making GDF Trap polypeptides or as directtherapeutic agents (e.g., in a gene therapy approach).

In certain aspects, the subject nucleic acids encoding GDF Trappolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NOs: 5, 25, 27, 30 and 31. Variant nucleotidesequences include sequences that differ by one or more nucleotidesubstitutions, additions or deletions, such as allelic variants; andwill, therefore, include coding sequences that differ from thenucleotide sequence of the coding sequence designated in SEQ ID NOs: 5,25, 27, 30 and 31.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NO: 5, 25, 27, 30 or 31. One of ordinaryskill in the art will appreciate that nucleic acid sequencescomplementary to SEQ ID NO: 5, 25, 27, 30 or 31, and variants of SEQ IDNO: 5, 25, 27, 30 or 31, are also within the scope of this invention. Infurther embodiments, the nucleic acid sequences of the invention can beisolated, recombinant, and/or fused with a heterologous nucleotidesequence, or in a DNA library.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NO: 5, 25, 27, 30 or 31,complement sequence of SEQ ID NO: 5, 25, 27, 30 or 31, or fragmentsthereof. As discussed above, one of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. For example, one could perform thehybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the invention providesnucleic acids which hybridize under low stringency conditions of 6×SSCat room temperature followed by a wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NO: 5, 25, 27, 30 or 31 due to degeneracy in the genetic codeare also within the scope of the invention. For example, a number ofamino acids are designated by more than one triplet. Codons that specifythe same amino acid, or synonyms (for example, CAU and CAC are synonymsfor histidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. In certain embodiments, the GDF Trappolypeptide will be encoded by an alternative nucleotide sequence.Alternative nucleotide sequences are degenerate with respect to thenative GDF Trap nucleic acid sequence but still encode for the samefusion protein. In certain embodiments, the GDF Trap having SEQ ID NO:26 is encoded by an alternative nucleic acid sequence comprising SEQ IDNO: 30. However, it is expected that DNA sequence polymorphisms that dolead to changes in the amino acid sequences of the subject proteins willexist among mammalian cells. One skilled in the art will appreciate thatthese variations in one or more nucleotides (up to about 3-5% of thenucleotides) of the nucleic acids encoding a particular protein mayexist among individuals of a given species due to natural allelicvariation. Any and all such nucleotide variations and resulting aminoacid polymorphisms are within the scope of this invention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the invention, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding a GDF Trap polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the GDF Trap polypeptide. Accordingly,the term regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding a GDF Trap polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant GDF Trap polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 1989) Chapters 16 and 17. In some instances, it may bedesirable to express the recombinant polypeptides by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject GDF Trap polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject GDF Trap polypeptides in cells propagated in culture,e.g., to produce proteins, including fusion proteins or variantproteins, for purification.

This invention also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 4, 5, 25,27, 30 or 31) for one or more of the subject GDF Trap polypeptides. Thehost cell may be any prokaryotic or eukaryotic cell. For example, a GDFTrap polypeptide of the invention may be expressed in bacterial cellssuch as E. coli, insect cells (e.g., using a baculovirus expressionsystem), yeast, or mammalian cells. Other suitable host cells are knownto those skilled in the art.

Accordingly, the present invention further pertains to methods ofproducing the subject GDF Trap polypeptides. For example, a host celltransfected with an expression vector encoding a GDF Trap polypeptidecan be cultured under appropriate conditions to allow expression of theGDF Trap polypeptide to occur. The GDF Trap polypeptide may be secretedand isolated from a mixture of cells and medium containing the GDF Trappolypeptide. Alternatively, the GDF Trap polypeptide may be retainedcytoplasmically or in a membrane fraction and the cells harvested, lysedand the protein isolated. A cell culture includes host cells, media andother byproducts. Suitable media for cell culture are well known in theart. The subject GDF Trap polypeptides can be isolated from cell culturemedium, host cells, or both, using techniques known in the art forpurifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for particularepitopes of the GDF Trap polypeptides. In a preferred embodiment, theGDF Trap polypeptide is a fusion protein containing a domain whichfacilitates its purification.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant GDF Trappolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified GDF Trap polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Screening Assays

In certain aspects, the present invention relates to the use of thesubject GDF Trap polypeptides (e.g., soluble variant ActRIIBpolypeptides) to identify compounds (agents) which are agonist orantagonists of ActRIIB polypeptides. Compounds identified through thisscreening can be tested to assess their ability to modulate red bloodcell, hemoglobin and/or reticulocyte levels in vivo or in vitro. Thesecompounds can be tested, for example, in animal models.

There are numerous approaches to screening for therapeutic agents forincreasing red blood cell or hemoglobin levels by targeting ActRIIBsignaling. In certain embodiments, high-throughput screening ofcompounds can be carried out to identify agents that perturbActRIIB-mediated effects on a selected cell line. In certainembodiments, the assay is carried out to screen and identify compoundsthat specifically inhibit or reduce binding of an ActRIIB polypeptide toits binding partner, such as an ActRIIB ligand (e.g., activin, Nodal,GDF8, GDF11 or BMP7). Alternatively, the assay can be used to identifycompounds that enhance binding of an ActRIIB polypeptide to its bindingpartner such as an ActRIIB ligand. In a further embodiment, thecompounds can be identified by their ability to interact with an ActRIIBpolypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 Daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between an ActRIIBpolypeptide and its binding partner (e.g., an ActRIIB ligand).

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified ActRIIB polypeptide which is ordinarily capable of binding toan ActRIIB ligand, as appropriate for the intention of the assay. To themixture of the compound and ActRIIB polypeptide is then added to acomposition containing an ActRIIB ligand. Detection and quantificationof ActRIIB/ActRIIB ligand complexes provides a means for determining thecompound's efficacy at inhibiting (or potentiating) complex formationbetween the ActRIIB polypeptide and its binding protein. The efficacy ofthe compound can be assessed by generating dose response curves fromdata obtained using various concentrations of the test compound.Moreover, a control assay can also be performed to provide a baselinefor comparison. For example, in a control assay, isolated and purifiedActRIIB ligand is added to a composition containing the ActRIIBpolypeptide, and the formation of ActRIIB/ActRIIB ligand complex isquantitated in the absence of the test compound. It will be understoodthat, in general, the order in which the reactants may be admixed can bevaried, and can be admixed simultaneously. Moreover, in place ofpurified proteins, cellular extracts and lysates may be used to render asuitable cell-free assay system.

Complex formation between the ActRIIB polypeptide and its bindingprotein may be detected by a variety of techniques. For instance,modulation of the formation of complexes can be quantitated using, forexample, detectably labeled proteins such as radiolabeled (e.g., ³²P,³⁵S, ¹⁴C or ³H), fluorescently labeled (e.g., FITC), or enzymaticallylabeled ActRIIB polypeptide or its binding protein, by immunoassay, orby chromatographic detection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between an ActRIIB polypeptide and its bindingprotein. Further, other modes of detection, such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between an ActRIIB polypeptideand its binding partner. See for example, U.S. Pat. No. 5,283,317;Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment,the present invention contemplates the use of reverse two hybrid systemsto identify compounds (e.g., small molecules or peptides) thatdissociate interactions between an ActRIIB polypeptide and its bindingprotein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, the subject compounds are identified by theirability to interact with an ActRIIB polypeptide. The interaction betweenthe compound and the ActRIIB polypeptide may be covalent ornon-covalent. For example, such interaction can be identified at theprotein level using in vitro biochemical methods, includingphoto-crosslinking, radiolabeled ligand binding, and affinitychromatography (Jakoby W B et al., 1974, Methods in Enzymology 46: 1).In certain cases, the compounds may be screened in a mechanism basedassay, such as an assay to detect compounds which bind to an ActRIIBpolypeptide. This may include a solid phase or fluid phase bindingevent. Alternatively, the gene encoding an ActRIIB polypeptide can betransfected with a reporter system (e.g., β-galactosidase, luciferase,or green fluorescent protein) into a cell and screened against thelibrary preferably by a high throughput screening or with individualmembers of the library. Other mechanism based binding assays may beused, for example, binding assays which detect changes in free energy.Binding assays can be performed with the target fixed to a well, bead orchip or captured by an immobilized antibody or resolved by capillaryelectrophoresis. The bound compounds may be detected usually usingcolorimetric or fluorescence or surface plasmon resonance.

5. Exemplary Therapeutic Uses

In certain embodiments, the GDF Trap polypeptides of the presentinvention can be used to increase red blood cell levels in mammals suchas rodents and primates, and particularly human patients. Additionally,as shown herein, GDF Trap polypeptides may be used in combination withEPO receptor activators to achieve an increase in red blood cells atlower dose ranges. This may be beneficial in reducing the knownoff-target effects and risks associated with high doses of EPO receptoractivators. In certain embodiments, the present invention providesmethods of treating or preventing anemia in an individual in needthereof by administering to the individual a therapeutically effectiveamount of a GDF Trap polypeptide or a combination (or concomitanttherapy) of a GDF Trap polypeptide and a EPO receptor activator. Thesemethods may be used for therapeutic and prophylactic treatments ofmammals, and particularly humans.

The GDF Trap polypeptides may be used in combination with EPO receptoractivators to reduce the required dose of these activators in patientsthat are susceptible to adverse effects of EPO. The primary adverseeffects of EPO are an excessive increase in the hematocrit or hemoglobinlevels and polycythemia. Elevated hematocrit levels can lead tohypertension (more particularly aggravation of hypertension) andvascular thrombosis. Other adverse effects of EPO which have beenreported, some of which related to hypertension, are headaches,influenza-like syndrome, obstruction of shunts, myocardial infarctionsand cerebral convulsions due to thrombosis, hypertensive encephalopathy,and red cell blood cell applasia (Singibarti, (1994) J. Clin Investig72(suppl 6), S36-S43; Horl et al. (2000) Nephrol Dial Transplant15(suppl 4), 51-56; Delanty et al. (1997) Neurology 49, 686-689; Bunn(2002) N Engl J Med 346(7), 522-523).

The rapid effect on red blood cell levels of the GDF Trap polypeptidesdisclosed herein indicate that these agents act by a different mechanismthan EPO. Accordingly, these antagonists may be useful for increasingred blood cell and hemoglobin levels in patients that do not respondwell to EPO. For example, a GDF Trap polypeptide may be beneficial for apatient in which administration of a normal to increased (>300IU/kg/week) dose of EPO does not result in the increase of hemoglobinlevel up to the target level. Patients with an inadequate EPO responseare found for all types of anemia, but higher numbers of non-respondershave been observed particularly frequently in patients with cancers andpatients with end-stage renal disease. An inadequate response to EPO canbe either constitutive (i.e. observed upon the first treatment with EPO)or acquired (e.g. observed upon repeated treatment with EPO).

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes prophylaxis of the named condition or amelioration orelimination of the condition once it has been established. In eithercase, prevention or treatment may be discerned in the diagnosis providedby a physician or other health care provider and the intended result ofadministration of the therapeutic agent.

As shown herein, GDF Trap polypeptides, optionally combined with an EPOreceptor activator, may be used to increase red blood cell, hemoglobinor reticulocyte levels in healthy individuals, and such GDF Trappolypeptides may be used in selected patient populations. Examples ofappropriate patient populations include those with undesirably low redblood cell or hemoglobin levels, such as patients having an anemia, andthose that are at risk for developing undesirably low red blood cell orhemoglobin levels, such as those patients that are about to undergomajor surgery or other procedures that may result in substantial bloodloss. In one embodiment, a patient with adequate red blood cell levelsis treated with a GDF Trap polypeptide to increase red blood celllevels, and then blood is drawn and stored for later use intransfusions.

GDF Trap polypeptides, optionally combined with an EPO receptoractivator, disclosed herein may be used to increase red blood celllevels in patients having an anemia. When observing hemoglobin levels inhumans, a level of less than normal for the appropriate age and gendercategory may be indicative of anemia, although individual variations aretaken into account. For example, a hemoglobin level of 12 g/dl isgenerally considered the lower limit of normal in the general adultpopulation. Potential causes include blood-loss, nutritional deficits,medication reaction, various problems with the bone marrow and manydiseases. More particularly, anemia has been associated with a varietyof disorders that include, for example, chronic renal failure,myelodysplastic syndrome, rheumatoid arthritis, bone marrowtransplantation. Anemia may also be associated with the followingconditions: solid tumors (e.g. breast cancer, lung cancer, coloncancer); tumors of the lymphatic system (e.g. chronic lymphocyteleukemia, non-Hodgkins and Hodgkins lymphomas); tumors of thehematopoietic system (e.g. leukemia, myelodysplastic syndrome, multiplemyeloma); radiation therapy; chemotherapy (e.g. platinum containingregimens); inflammatory and autoimmune diseases, including, but notlimited to, rheumatoid arthritis, other inflammatory arthritides,systemic lupus erythematosis (SLE), acute or chronic skin diseases (e.g.psoriasis), inflammatory bowel disease (e.g. Crohn's disease andulcerative colitis); acute or chronic renal disease or failure includingidiopathic or congenital conditions; acute or chronic liver disease;acute or chronic bleeding; situations where transfusion of red bloodcells is not possible due to patient allo- or auto-antibodies and/or forreligious reasons (e.g. some Jehovah's Witnesses); infections (e.g.malaria, osteomyelitis); hemoglobinopathies, including, for example,sickle cell disease, thalassemias; drug use or abuse, e.g. alcoholmisuse; pediatric patients with anemia from any cause to avoidtransfusion; and elderly patients or patients with underlyingcardiopulmonary disease with anemia who cannot receive transfusions dueto concerns about circulatory overload.

Myelodysplastic syndrome (MDS) is a diverse collection of hematologicalconditions characterized by ineffective production of myeloid bloodcells and risk of transformation to acute mylogenous leukemia. In MDSpatients, blood stem cells do not mature into healthy red blood cells,white blood cells, or platelets. MDS disorders include, for example,refractory anemia, refractory anemia with ringed sideroblasts,refractory anemia with excess blasts, refractory anemia with excessblasts in transformation, refractory cytopenia with multilineagedysplasia, and myelodysplastic syndrome associated with an isolated 5qchromosome abnormality. As these disorders manifest as irreversibledefects in both quantity and quality of hematopoietic cells, most MDSpatients are afflicted with chronic anemia. Therefore, MDS patientseventually require blood transfusions and/or treatment with growthfactors (e.g., erythropoietin or G-CSF) to increase red blood celllevels. However, many MDS patients develop side-effect due to frequencyof such therapies. For example, patients who receive frequent red bloodcell transfusion can have tissue and organ damage from the buildup ofextra iron. As demonstrated in the Examples below, GDF Trap polypeptideswere used to treat anemia in a mouse model of MDS. Accordingly, GDF Trappolypeptides disclosed herein may be used to treat patients having MDS.In certain embodiments, patients suffering from MDS may be treated usinga combination of a GDF Trap polypeptide in combination with an EPOreceptor activator. In other embodiments, patient suffering from MDS maybe treated using a combination of a GDF Trap polypeptide and one or moreadditional therapeutic agents for treating MDS including, for example,thalidomide, lenalidomide, azacitadine, decitabine, erythropoietins,deferoxamine, antihymocyte globulin, filgrastrim (G-CSF) and anerythropoietin signaling pathway agonist.

GDF Trap polypeptides, optionally combined with an EPO receptoractivator, would be appropriate for treating anemias ofhypoproliferative bone marrow, which are typically associated withlittle change in red blood cell (RBC) morphology. Hypoproliferativeanemias include: 1) anemia of chronic disease, 2) anemia of kidneydisease, and 3) anemia associated with hypometabolic states. In each ofthese types, endogenous erythropoietin levels are inappropriately lowfor the degree of anemia observed. Other hypoproliferative anemiasinclude: 4) early-stage iron-deficient anemia, and 5) anemia caused bydamage to the bone marrow. In these types, endogenous erythropoietinlevels are appropriately elevated for the degree of anemia observed.

The most common type is anemia of chronic disease, which encompassesinflammation, infection, tissue injury, and conditions such as cancer,and is distinguished by both low erythropoietin levels and an inadequateresponse to erythropoietin in the bone marrow (Adamson, 2008, Harrison'sPrinciples of Internal Medicine, 17th ed.; McGraw Hill, New York, pp628-634). Many factors can contribute to cancer-related anemia. Some areassociated with the disease process itself and the generation ofinflamatory cytokines such as interleukin-1, interferon-gamma, and tumornecrosis factor (Bron et al., 2001, Semin Oncol 28(Suppl 8):1-6). Amongits effects, inflammation induces the key iron-regulatory peptidehepcidin, thereby inhibiting iron export from macrophages and generallylimiting iron availability for erythropoiesis (Ganz, 2007, J Am SocNephrol 18:394-400). Blood loss through various routes can alsocontribute to cancer-related anemia. The prevalence of anemia due tocancer progression varies with cancer type, ranging from 5% in prostatecancer up to 90% in multiple myeloma. Cancer-related anemia has profoundconsequences for patients, including fatigue and reduced quality oflife, reduced treatment efficacy, and increased mortality.

Chronic kidney disease is associated with hypoproliferative anemia thatvaries in severity with the degree of renal impairment. Such anemia isprimarily due to inadequate production of erythropoietin and reducedsurvival of red blood cells. Chronic kidney disease usually proceedsgradually over a period of years or decades to end-stage (Stage-5)disease, at which point dialysis or kidney transplantation is requiredfor patient survival. Anemia often develops early in this process andworsens as disease progresses. The clinical consequences of anemia ofkidney disease are well-documented and include development of leftventricular hypertrophy, impaired cognitive function, reduced quality oflife, and altered immune function (Levin et al., 1999, Am J Kidney Dis27:347-354; Nissenson, 1992, Am J Kidney Dis 20(Suppl 1):21-24; Revickiet al., 1995, Am J Kidney Dis 25:548-554; Gafter et al., 1994, KidneyInt 45:224-231). As demonstrated by the Applicants in a mouse model ofchronic kidney disease (see Example below), a GDF Trap polypeptide,optionally combined with an EPO receptor activator, can be used to treatanemia of kidney disease.

Many conditions resulting in a hypometabolic rate can produce amild-to-moderate hypoproliferative anemia. Among such conditions areendocrine deficiency states. For example, anemia can occur in Addison'sdisease, hypothyroidism, hyperparathyroidism, or males who are castratedor treated with estrogen. Mild-to-moderate anemia can also occur withreduced dietary intake of protein, a condition particularly prevalent inthe elderly. Finally, anemia can develop in patients with chronic liverdisease arising from nearly any cause (Adamson, 2008, Harrison'sPrinciples of Internal Medicine, 17th ed.; McGraw Hill, New York, pp628-634).

Anemia resulting from acute blood loss of sufficient volume, such asfrom trauma or postpartum hemorrhage, is known as acute post-hemorrhagicanemia. Acute blood loss initially causes hypovolemia without anemiasince there is proportional depletion of RBCs along with other bloodconstituents. However, hypovolemia will rapidly trigger physiologicmechanisms that shift fluid from the extravascular to the vascularcompartment, which results in hemodilution and anemia. If chronic, bloodloss gradually depletes body iron stores and eventually leads to irondeficiency. As demonstrated by the Applicants in a mouse model (seeExample below), a GDF Trap polypeptide, optionally combined with an EPOreceptor activator, can be used to speed recovery from anemia of acuteblood loss.

Iron-deficiency anemia is the final stage in a graded progression ofincreasing iron deficiency which includes negative iron balance andiron-deficient erythropoiesis as intermediate stages. Iron deficiencycan result from increased iron demand, decreased iron intake, orincreased iron loss, as exemplified in conditions such as pregnancy,inadequate diet, intestinal malabsorption, acute or chronicinflammation, and acute or chronic blood loss. With mild-to-moderateanemia of this type, the bone marrow remains hypoproliferative, and RBCmorphology is largely normal; however, even mild anemia can result insome microcytic hypochromic RBCs, and the transition to severeiron-deficient anemia is accompanied by hyperproliferation of the bonemarrow and increasingly prevalent microcytic and hypochromic RBCs(Adamson, 2008, Harrison's Principles of Internal Medicine, 17th ed.;McGraw Hill, New York, pp 628-634). Appropriate therapy foriron-deficiency anemia depends on its cause and severity, with oral ironpreparations, parenteral iron formulations, and RBC transfusion as majorconventional options. A GDF Trap polypeptide, optionally combined withan EPO receptor activator, could be used to treat chroniciron-deficiency anemias alone or in combination with conventionaltherapeutic approaches, particularly to treat anemias of multifactorialorigin.

Hypoproliferative anemias can result from primary dysfunction or failureof the bone marrow, instead of dysfunction secondary to inflammation,infection, or cancer progression. Prominent examples would bemyelosuppression caused by cancer chemotherapeutic drugs or cancerradiation therapy. A broad review of clinical trials found that mildanemia can occur in 100% of patients after chemotherapy, while moresevere anemia can occur in up to 80% of such patients (Groopman et al.,1999, J Natl Cancer Inst 91:1616-1634). Myelosuppressive drugsinclude: 1) alkylating agents such as nitrogen mustards (e.g.,melphalan) and nitrosoureas (e.g., streptozocin); 2) antimetabolitessuch as folic acid antagonists (e.g., methotrexate), purine analogs(e.g., thioguanine), and pyrimidine analogs (e.g., gemcitabine); 3)cytotoxic antibotics such as anthracyclines (e.g., doxorubicin); 4)kinase inhibitors (e.g., gefitinib); 5) mitotic inhibitors such astaxanes (e.g., paclitaxel) and vinca alkaloids (e.g., vinorelbine); 6)monoclonal antibodies (e.g., rituximab); and 7) topoisomerase inhibitors(e.g., topotecan and etoposide). As demonstrated in a mouse model ofchemotherapy-induced anemia (see Example below), a GDF Trap polypeptide,optionally combined with an EPO receptor activator, can be used to treatanemia caused by chemotherapeutic agents and/or radiation therapy.

GDF Trap polypeptides, optionally combined with an EPO receptoractivator, would also be appropriate for treating anemias of disorderedRBC maturation, which are characterized in part by undersized(microcytic), oversized (macrocytic), misshapen, or abnormally colored(hypochromic) RBCs.

Patients may be treated with a dosing regimen intended to restore thepatient to a target hemoglobin level, usually between about 10 g/dl andabout 12.5 g/dl, and typically about 11.0 g/dl (see also Jacobs et al.(2000) Nephrol Dial Transplant 15, 15-19), although lower target levelsmay cause fewer cardiovascular side effects. Alternatively, hematocritlevels (percentage of the volume of a blood sample occupied by thecells) can be used as a measure for the condition of red blood cells.Hematocrit levels for healthy individuals range from 41 to 51% for adultmales and from 35 to 45% for adult females. Target hematocrit levels areusually around 30-33%. Moreover, hemoglobin/hematocrit levels vary fromperson to person. Thus, optimally, the target hemoglobin/hematocritlevel can be individualized for each patient.

In certain embodiments, the present invention provides methods formanaging a patient that has been treated with, or is a candidate to betreated with, a GDF Trap polypeptide by measuring one or morehematologic parameters in the patient. The hematologic parameters may beused to evaluate appropriate dosing for a patient who is a candidate tobe treated with a GDF Trap polypeptide, to monitor the hematologicparameters during treatment with a GDF Trap polypeptide, to evaluatewhether to adjust the dosage during treatment with a GDF Trappolypeptide, and/or to evaluate an appropriate maintenance dose of a GDFTrap polypeptide. If one or more of the hematologic parameters areoutside the normal level, dosing with a GDF Trap polypeptide may bereduced, delayed or terminated.

Hematologic parameters that may be measured in accordance with themethods provided herein include, for example, red blood cell levels,blood pressure, iron stores, and other agents found in bodily fluidsthat correlate with increased red blood cell levels, using artrecognized methods. Such parameters may be determined using a bloodsample from a patient. Increases in red blood cell levels, hemoglobinlevels, and/or hematocrit levels may cause increases in blood pressure.

In one embodiment, if one or more hematologic parameters are outside thenormal range, or on the high side of normal, in a patient who is acandidate to be treated with a GDF Trap polypeptide then onset ofadministration of the GDF Trap polypeptide may be delayed until thehematologic parameters have returned to a normal or acceptable leveleither naturally or via therapeutic intervention. For example, if acandidate patient is hypertensive or prehypertensive, then the patientmay be treated with a blood pressure lowering agent in order to reducethe patient's blood pressure. Any blood pressure lowering agentappropriate for the individual patient's condition may be usedincluding, for example, diuretics, adrenergic inhibitors (includingalpha blockers and beta blockers), vasodilators, calcium channelblockers, angiotensin-converting enzyme (ACE) inhibitors, or angiotensinII receptor blockers. Blood pressure may alternatively be treated usinga diet and exercise regimen. Similarly, if a candidate patient has ironstores that are lower than normal, or on the low side of normal, thenthe patient may be treated with an appropriate regimen of diet and/oriron supplements until the patient's iron stores have returned to anormal or acceptable level. For patients having higher than normal redblood cell levels and/or hemoglobin levels, then administration of theGDF Trap polypeptide may be delayed until the levels have returned to anormal or acceptable level.

In certain embodiments, if one or more hematologic parameters areoutside the normal range, or on the high side of normal, in a patientwho is a candidate to be treated with a GDF Trap polypeptide then theonset of administration may be not be delayed. However, the dosageamount or frequency of dosing of the GDF Trap polypeptide may be set atan amount that would reduce the risk of an unacceptable increase in thehematologic parameters arising upon administration of the GDF Trappolypeptide. Alternatively, a therapeutic regimen may be developed forthe patient that combines a GDF Trap polypeptide with a therapeuticagent that addresses the undesirable level of the hematologic parameter.For example, if the patient has elevated blood pressure, then atherapeutic regimen involving administration of a GDF Trap polypeptideand a blood pressure lowering agent may be designed. For a patienthaving lower than desired iron stores, a therapeutic regimen of a GDFTrap polypeptide and iron supplementation may be developed.

In one embodiment, baseline parameter(s) for one or more hematologicparameters may be established for a patient who is a candidate to betreated with a GDF Trap polypeptide and an appropriate dosing regimenestablish for that patient based on the baseline value(s).

Alternatively, established baseline parameters based on a patient'smedical history could be used to inform an appropriate GDF Trappolypeptide dosing regimen for a patient. For example, if a healthypatient has an established baseline blood pressure reading that is abovethe defined normal range it may not be necessary to bring the patient'sblood pressure into the range that is considered normal for the generalpopulation prior to treatment with the GDF Trap polypeptide. A patient'sbaseline values for one or more hematologic parameters prior totreatment with a GDF Trap polypeptide may also be used as the relevantcomparative values for monitoring any changes to the hematologicparameters during treatment with the GDF Trap polypeptide.

In certain embodiments, one or more hematologic parameters are measuredin patients who are being treated with a GDF Trap polypeptide. Thehematologic parameters may be used to monitor the patient duringtreatment and permit adjustment or termination of the dosing with theGDF Trap polypeptide or additional dosing with another therapeuticagent. For example, if administration of a GDF Trap polypeptide resultsin an increase in blood pressure, red blood cell level, or hemoglobinlevel, or a reduction in iron stores, then the dose of the GDF Trappolypeptide may be reduced in amount or frequency in order to decreasethe effects of the GDF Trap polypeptide on the one or more hematologicparameters. If administration or a GDF Trap polypeptide results in achange in one or more hematologic parameters that is adverse to thepatient, then the dosing of the GDF Trap polypeptide may be terminatedeither temporarily, until the hematologic parameter(s) return to anacceptable level, or permanently. Similarly, if one or more hematologicparameters are not brought within an acceptable range after reducing thedose or frequency of administration of the GDF Trap polypeptide then thedosing may be terminated. As an alternative, or in addition to, reducingor terminating the dosing with the GDF Trap polypeptide, the patient maybe dosed with an additional therapeutic agent that addresses theundesirable level in the hematologic parameter(s), such as, for example,a blood pressure lowering agent or an iron supplement. For example, if apatient being treated with a GDF Trap polypeptide has elevated bloodpressure, then dosing with the GDF Trap polypeptide may continue at thesame level and a blood pressure lowering agent is added to the treatmentregimen, dosing with the GDF Trap polypeptide may be reduce (e.g., inamount and/or frequency) and a blood pressure lowering agent is added tothe treatment regimen, or dosing with the GDF Trap polypeptide may beterminated and the patient may be treated with a blood pressure loweringagent.

In certain embodiments, patients being treated with a GDF Trappolypeptide, or candidate patients to be treated with a GDF Trappolypeptide, are patients in need of muscle growth, such as patientssuffering from, or at risk of developing, a neuromuscular disorder ormusculogenerative disorder. For example, patients or candidate patientsmay be suffering from, or at risk for developing, Lou Gehrig's disease(ALS), cancer anorexia-cachexia syndrome, muscular dystrophy, muscleatrophy, congestive obstructive pulmonary disease (and muscle wastingassociated with COPD), muscle wasting syndrome, sarcopenia, or cachexia.Muscular dystrophy refers to a group of degenerative muscle diseasescharacterized by gradual weakening and deterioration of skeletal musclesand sometimes the heart and respiratory muscles. Exemplary musculardystrophies that can be treated with a regimen including the subject GDFTrap polypeptides include: Duchenne Muscular Dystrophy (DMD), BeckerMuscular Dystrophy (BMD), Emery-Dreifuss Muscular Dystrophy (EDMD),Limb-Girdle Muscular Dystrophy (LGMD), Facioscapulohumeral MuscularDystrophy (FSH or FSHD) (also known as Landouzy-Dejerine), MyotonicDystrophy (MMD) (also known as Steinert's Disease), OculopharyngealMuscular Dystrophy (OPMD), Distal Muscular Dystrophy (DD), CongenitalMuscular Dystrophy (CMD).

6. Pharmaceutical Compositions

In certain embodiments, compounds (e.g., GDF Trap polypeptides) of thepresent invention are formulated with a pharmaceutically acceptablecarrier. For example, a GDF Trap polypeptide can be administered aloneor as a component of a pharmaceutical formulation (therapeuticcomposition). The subject compounds may be formulated for administrationin any convenient way for use in human or veterinary medicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition systemically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Therapeutically useful agents other than the GDF Trap polypeptideswhich may also optionally be included in the composition as describedabove, may be administered simultaneously or sequentially with thesubject compounds (e.g., GDF Trap polypeptides) in the methods of theinvention.

Typically, compounds will be administered parenterally. Pharmaceuticalcompositions suitable for parenteral administration may comprise one ormore GDF Trap polypeptides in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

Further, the composition may be encapsulated or injected in a form fordelivery to a target tissue site (e.g., bone marrow). In certainembodiments, compositions of the present invention may include a matrixcapable of delivering one or more therapeutic compounds (e.g., GDF Trappolypeptides) to a target tissue site (e.g., bone marrow), providing astructure for the developing tissue and optimally capable of beingresorbed into the body. For example, the matrix may provide slow releaseof the GDF Trap polypeptides. Such matrices may be formed of materialspresently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., GDF Trap polypeptides).The various factors include, but are not limited to, the patient's redblood cell count, hemoglobin level or other diagnostic assessments, thedesired target red blood cell count, the patient's age, sex, and diet,the severity of any disease that may be contributing to a depressed redblood cell level, time of administration, and other clinical factors.The addition of other known growth factors to the final composition mayalso affect the dosage. Progress can be monitored by periodic assessmentof red blood cell and hemoglobin levels, as well as assessments ofreticulocyte levels and other indicators of the hematopoietic process.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of GDF Trap polypeptides. Such therapy wouldachieve its therapeutic effect by introduction of the GDF Trappolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of GDF Trap polynucleotide sequences can beachieved using a recombinant expression vector such as a chimeric virusor a colloidal dispersion system. Preferred for therapeutic delivery ofGDF Trap polynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or an RNA virus suchas a retrovirus. The retroviral vector may be a derivative of a murineor avian retrovirus. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). Anumber of additional retroviral vectors can incorporate multiple genes.All of these vectors can transfer or incorporate a gene for a selectablemarker so that transduced cells can be identified and generated.Retroviral vectors can be made target-specific by attaching, forexample, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody. Those of skill in the art willrecognize that specific polynucleotide sequences can be inserted intothe retroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing the GDF Trappolynucleotide.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for GDF Trap polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1. Generation of a GDF Trap

Applicants constructed a GDF Trap as follows. A polypeptide having amodified extracellular domain of ActRIIB with greatly reduced activin Abinding relative to GDF11 and/or myostatin (as a consequence of aleucine-to-aspartate substitution at position 79 in SEQ ID NO: 1) wasfused to a human or mouse Fc domain with a minimal linker (three glycineamino acids) in between. The constructs are referred to as ActRIIB(L79D20-134)-hFc and ActRIIB(L79D 20-134)-mFc, respectively. Alternativeforms with a glutamate rather than an aspartate at position 79 performedsimilarly (L79E). Alternative forms with an alanine rather than a valineat position 226 with respect to SEQ ID NO: 7, below were also generatedand performed equivalently in all respects tested. The aspartate atposition 79 (relative to SEQ ID NO: 1, or position 60 relative to SEQ IDNO: 7) is highlighted in bold below. The valine at position 226 relativeto SEQ ID NO: 7 is also highlighted in bold below.

The GDF Trap ActRIIB(L79D 20-134)-hFc is shown below as purified fromCHO cell lines (SEQ ID NO: 7).

GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSG

EAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY

TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The ActRIIB-derived portion of the GDF Trap has an amino acid sequenceset forth below (SEQ ID NO: 32), and that portion could be used as amonomer or as a non-Fc fusion protein as a monomer, dimer or greaterorder complex.

(SEQ ID NO: 32) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLH

QVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT

The GDF Trap protein was expressed in CHO cell lines. Three differentleader sequences were considered:

(i) Honey bee melittin (HBML): (SEQ ID NO: 8) MKFLVNVALVFMVVYISYIYA(ii) Tissue Plasminogen Activator (TPA): (SEQ ID NO: 9)MDAMKRGLCCVLLLCGAVFVSP (iii) Native: (SEQ ID NO: 10)MTAPWVALALLWGSLCAGS.

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence:

(SEQ ID NO: 11) MDAMKRGLCCVLLLCGAVFVSPGASGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence (SEQID NO:12):

A TGGATGCAAT GAAGAGAGGG CTCTGCTGTG TGCTGCTGCTGTGTGGAGCA GTCTTCGTTT CGCCCGGCGC CTCTGGGCGTGGGGAGGCTG AGACACGGGA GTGCATCTAC TACAACGCCAACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTGGAGCGCTGCGAAGGC GAGCAGGACA AGCGGCTGCA CTGCTACGCCTCCTGGCGCA ACAGCTCTGG CACCATCGAG CTCGTGAAGAAGGGCTGCTG GGACGATGAC TTCAACTGCT ACGATAGGCAGGAGTGTGTG GCCACTGAGG AGAACCCCCA GGTGTACTTCTGCTGCTGTG AAGGCAACTT CTGCAACGAG CGCTTCACTCATTTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTACGAGCCACCCCCGACA GCCCCCACCG GTGGTGGAAC TCACACATGCCCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAGTCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGATCTCCCGGACC CCTGAGGTCA CATGCGTGGT GGTGGACGTGAGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGGACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGAGGAGCAGTAC AACAGCACGT ACCGTGTGGT CAGCGTCCTCACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACAAGTGCAAGGT CTCCAACAAA GCCCTCCCAG TCCCCATCGAGAAAACCATC TCCAAAGCCA AAGGGCAGCC CCGAGAACCACAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCAAGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTATCCCAGCGAC ATCGCCGTGG AGTGGGAGAG CAATGGGCAGCCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACTCCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGACAAGAGCAGG TGGCAGCAGG GGAACGTCTT CTCATGCTCCGTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGA GCCTCTCCCT GTCTCCGGGT AAATGA

Purification could be achieved by a series of column chromatographysteps, including, for example, three or more of the following, in anyorder: protein A chromatography, Q sepharose chromatography,phenylsepharose chromatography, size exclusion chromatography, andcation exchange chromatography. The purification could be completed withviral filtration and buffer exchange. In an example of a purificationscheme, the cell culture medium is passed over a protein A column,washed in 150 mM Tris/NaCl (pH 8.0), then washed in 50 mM Tris/NaCl (pH8.0) and eluted with 0.1 M glycine, pH 3.0. The low pH eluate is kept atroom temperature for 30 minutes as a viral clearance step. The eluate isthen neutralized and passed over a Q sepharose ion exchange column andwashed in 50 mM Tris pH 8.0, 50 mM NaCl, and eluted in 50 mM Tris pH8.0, with an NaCl concentration between 150 mM and 300 mM. The eluate isthen changed into 50 mM Tris pH 8.0, 1.1 M ammonium sulfate and passedover a phenyl sepharose column, washed, and eluted in 50 mM Tris pH 8.0with ammonium sulfate between 150 and 300 mM. The eluate is dialyzed andfiltered for use.

Additional GDF Traps (ActRIIB-Fc fusion proteins modified so as toreduce the ratio of activin A binding relative to myostatin or GDF11)are described in PCT/US2008/001506 and WO 2006/012627, incorporated byreference herein.

Example 2. Bioassay for GDF-11 and Activin-Mediated Signaling

An A-204 Reporter Gene Assay was used to evaluate the effects ofActRIIB-Fc proteins and GDF Traps on signaling by GDF-11 and Activin A.Cell line: Human Rhabdomyosarcoma (derived from muscle). Reportervector: pGL3(CAGA)12 (Described in Dennler et al, 1998, EMBO 17:3091-3100). The CAGA12 motif is present in TGF-Beta responsive genes(PAI-1 gene), so this vector is of general use for factors signalingthrough Smad2 and 3.

Day 1: Split A-204 cells into 48-well plate.

Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 orpGL3(CAGA)12(10 ug)+pRLCMV (1 ug) and Fugene.

Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors need to bepreincubated with Factors for 1 hr before adding to cells. 6 hrs later,cells rinsed with PBS, and lyse cells.

This is followed by a Luciferase assay. In the absence of anyinhibitors, Activin A showed 10 fold stimulation of reporter geneexpression and an ED50 ˜2 ng/ml. GDF-11: 16 fold stimulation, ED50: ˜1.5ng/ml.

ActRIIB(20-134) is a potent inhibitor of activin, GDF-8 and GDF-11activity in this assay. Variants were tested in this assay as well.

Example 3. GDF-11 Inhibition by N-Terminal and C-Terminal Truncations

Variants of ActRIIB(20-134)-hFc with truncations at the N-terminusand/or C-terminus were generated and tested for activity as inhibitorsof GDF-11 and activin. The activities are shown below (as measured inconditioned media):

C-Terminal ActRIIB-hFc Truncations:

IC50 (ng/mL) GDF-11 Activin ActRIIB(20-134)-hFc 45 22ActRIIB(20-132)-hFc 87 32 ActRIIB(20-131)-hFc 120 44 ActRIIB(20-128)-hFc130 158

As can be seen, truncations of three (ending with . . . PPT), six(ending with . . . YEP) or more amino acids at the C-terminus causes athreefold or greater decrease in the activity of the molecule. Thetruncation of the final 15 amino acids of the ActRIIB portion causes agreater loss of activity (see WO2006/012627).

Amino terminal truncations were made in the background of anActRIIB(20-131)-hFc protein. The activities are shown below (as measuredin conditioned media):

N-Terminal ActRIIB-hFc Truncations:

IC50 (ng/mL) GDF-11 Activin ActRIIB(20-131)-hFc 183 201 (GRG . . .)ActRIIB(21-131)-hFc 121 325 (RGE . . .) ActRIIB(22-131)-hFc 71 100 (GEA. . .) ActRIIB(23-131)-hFc 60 43 (EAE . . .) ActRIIB(24-131)-hFc 69 105(AET . . .)

Accordingly, truncations of two, three or four amino acids from theN-terminus lead to the production of a more active protein than theversions with a full-length extracellular domain. Additional experimentsshow that a truncation of five amino acids, ActRIIB(25-131)-hFc hasactivity equivalent to the untruncated form, and additional deletions atthe N-terminus continue to degrade the activity of the protein.Therefore, optimal constructs will have a C-terminus ending betweenamino acid 133-134 of SEQ ID NO: 1 and an N-terminus beginning at aminoacids 22-24 of SEQ ID NO: 1. An N-terminus corresponding to amino acids21 or 25 will give activity that is similar to the ActRIIB(20-134)-hFcconstruct. These truncations may also be used in the context of GDFTraps, such as an L79D or L79E variant.

Example 4. ActRIIB-Fc Variants, Cell-Based Activity

Activity of ActRIIB-Fc proteins and GDF Traps was tested in a cell basedassay, as described above. Results are summarized in the table below.Some variants were tested in different C-terminal truncation constructs.As discussed above, truncations of five or fifteen amino acids causedreduction in activity. The GDF Traps (L79D and L79E variants) showedsubstantial loss of activin binding while retaining almost wild-typeinhibition of GDF-11.

Soluble ActRIIB-Fc Binding to GDF11 and Activin A:

Portion of ActRIIB (corresponds GDF11 Activin ActRIIB-Fc to amino acidsof Inhibition Inhibition Variations SEQ ID NO: 1) Activity Activity R6420-134 +++ +++ (approx. (approx. 10⁻⁸ M K_(I)) 10⁻⁸ M K_(I)) A6420-134 + + (approx. (approx. 10⁻⁶ M K_(I)) 10⁻⁶ M K_(I)) R64 20-129 ++++++ R64 K74A 20-134 ++++ ++++ R64 A24N 20-134 +++ +++ R64 A24N 20-119 ++++ R64 A24N K74A 20-119 + + R64 L79P 20-134 + + R64 L79P K74A 20-134 + +R64 L79D 20-134 +++ + R64 L79E 20-134 +++ + R64K 20-134 +++ +++ R64K20-129 +++ +++ R64 P129S P130A 20-134 +++ +++ R64N 20-134 + + + Pooractivity (roughly 1 × 10⁻⁶ K_(I)) ++ Moderate activity (roughly 1 × 10⁻⁷K_(I)) +++ Good (wild-type) activity (roughly 1 × 10⁻⁸ K_(I)) ++++Greater than wild-type activity

Several variants have been assessed for serum half-life in rats.ActRIIB(20-134)-Fc has a serum half-life of approximately 70 hours.ActRIIB(A24N 20-134)-Fc has a serum half-life of approximately 100-150hours. The A24N variant has activity in the cell-based assay (above) andin vivo assays (below) that are equivalent to the wild-type molecule.Coupled with the longer half-life, this means that over time an A24Nvariant will give greater effect per unit of protein than the wild-typemolecule. The A24N variant, and any of the other variants tested above,may be combined with the GDF Trap molecules, such as the L79D or L79Evariants.

Example 5. GDF-11 and Activin A Binding

Binding of certain ActRIIB-Fc proteins and GDF Traps to ligands wastested in a BiaCore™ assay.

The ActRIIB-Fc variants or wild-type protein were captured onto thesystem using an anti-hFc antibody. Ligands were injected and flowed overthe captured receptor proteins.

Results are summarized in the tables, below.

Ligand Binding Specificity IIB Variants.

Kon Koff KD Protein (1/Ms) (1/s) (M) GDF11 ActRIIB(20-134)-hFc 1.34e−61.13e−4 8.42e−11 ActRIIB(A24N 20-134)-hFc 1.21e−6 6.35e−5 5.19e−11ActRIIB(L79D 20-134)-hFc  6.7e−5 4.39e−4 6.55e−10 ActRIIB(L79E20-134)-hFc  3.8e−5 2.74e−4 7.16e−10 ActRIIB(R64K 20-134)-hFc 6.77e−52.41e−5 3.56e−11 GDF8 ActRIIB(20-134)-hFc 3.69e−5 3.45e−5 9.35e−11ActRIIB(A24N 20-134)-hFc ActRIIB(L79D 20-134)-hFc 3.85e−5  8.3e−42.15e−9  ActRIIB(L79E 20-134)-hFc 3.74e−5   9e−4 2.41e−9  ActRIIB(R64K20-134)-hFc 2.25e−5 4.71e−5  2.1e−10 ActRIIB(R64K 20-129)-hFc 9.74e−42.09e−4 2.15e−9  ActRIIB(P129S, P130R 20-134)- 1.08e−5  1.8e−4 1.67e−9 hFc ActRIIB(K74A 20-134)-hFc  2.8e−5 2.03e−5 7.18e−11 ActivinAActRIIB(20-134)-hFc 5.94e6  1.59e−4 2.68e−11 ActRIIB(A24N 20-134)-hFc3.34e6  3.46e−4 1.04e−10 ActRIIB(L79D 20-134)-hFc Low bindingActRIIB(L79E 20-134)-hFc Low binding ActRIIB(R64K 20-134)-hFc 6.82e6 3.25e−4 4.76e−11 ActRIIB(R64K 20-129)-hFc 7.46e6  6.28e−4 8.41e−11ActRIIB(P129S, P130R 20-134)- 5.02e6  4.17e−4 8.31e−11 hFc

These data confirm the cell based assay data, demonstrating that theA24N variant retains ligand-binding activity that is similar to that ofthe ActRIIB(20-134)-hFc molecule, and that the L79D or L79E moleculeretains myostatin and GDF11 binding but shows markedly decreased(non-quantifiable) binding to Activin A.

Other variants have been generated and tested, as reported inWO2006/012627 (incorporated herein by reference in its entirety) seee.g., pp. 59-60, using ligands coupled to the device and flowingreceptor over the coupled ligands. Notably, K74Y, K74F, K74I (andpresumably other hydrophobic substitutions at K74, such as K74L), andD80I, cause a decrease in the ratio of Activin A binding to GDF11binding, relative to the wild-type K74 molecule. A table of data withrespect to these variants is reproduced below:

Soluble ActRIIB-Fc Variants Binding to GDF11 and Activin A (BiaCoreAssay)

ActRIIB ActA GDF11 WT (64A) KD = 1.8e−7M KD = 2.6e−7M (+) (+) WT (64R)na KD = 8.6e−8M (+++) +15tail KD ~2.6e−8M KD = 1.9e−8M (+++) (++++)E37A * * R40A − − D54A − * K55A ++ * R56A * * K74A KD = 4.35e−9M KD =5.3e−9M +++++ +++++ K74Y * −− K74F * −− K74I * −− W78A * * L79A + *D80K * * D80R * * D80A * * D80F * * D80G * * D80M * * D80N * * D80I * −−F82A ++ − * No observed binding −− <⅕ WT binding − ~½ WT binding + WT ++<2× increased binding +++ ~5× increased binding ++++ ~10× increasedbinding +++++ ~40× increased binding

Example 6. ActRIIB-hFc Stimulates Erythropoiesis in Non-Human Primates

ActRIIB(20-134)-hFc (IgG1) was administered once a week for 1 month tomale and female cynomolgus monkeys by subcutaneous injection.Forty-eight cynomolgus monkeys (24/sex) were assigned to one of fourtreatment groups (6 animals/sex/group) and were administeredsubcutaneous injections of either vehicle or ActRIIB-hFc at 3, 10, or 30mg/kg once weekly for 4 weeks (total of 5 doses). Parameters evaluatedincluded general clinical pathology (hematology, clinical chemistry,coagulation, and urinalysis). ActRIIB-hFc caused statisticallysignificant elevated mean absolute reticulocyte values by day 15 intreated animals. By day 36, ActRIM-hFc caused several hematologicalchanges, including elevated mean absolute reticulocyte and red bloodcell distribution width values and lower mean corpuscular hemoglobinconcentration. All treated groups and both sexes were affected. Theseeffects are consistent with a positive effect of ActRIIB-hFc on therelease of immature reticulocytes from the bone marrow. This effect wasreversed after drug was washed out of the treated animals (by study day56). Accordingly, we conclude that ActRIIB-hFc stimulateserythropoiesis.

Example 7. ActRIIB-mFc Promotes Aspects of Erythropoiesis in Mice byStimulation of Splenic Erythropoietic Activities

In this study the effects of the in vivo administration ofActRIIB(20-134)-mFc on the frequency of hematopoietic progenitors inbone marrow and spleen was analyzed. One group of C57BL/6 mice wasinjected with PBS as a control and a second group of mice administeredtwo doses of ActRIIB-mFc at 10 mg/kg and both groups sacrificed after 8days. Peripheral blood was used to perform complete blood counts andfemurs and spleens were used to perform in vitro clonogenic assays toassess the lymphoid, erythroid and myeloid progenitor cell content ineach organ. In the brief time frame of this study, no significantchanges were seen in red blood cell, hemoglobin or white blood celllevels in treated mice. In the femurs there was no difference in thenucleated cell numbers or progenitor content between the control andtreated groups. In the spleens, the compound treated group experienced astatistically significant increase in the mature erythroid progenitor(CFU-E) colony number per dish, frequency and total progenitor numberper spleen. In addition, and increase was seen in the number of myeloid(CFU-GM), immature erythroid (BFU-E) and total progenitor number perspleen.

Animals:

Sixteen C57BL/6 female mice 6-8 weeks of age were used in the study.Eight mice were injected subcutaneously with test compound ActRIIB-mFcat days 1 and 3 at a dose of 10 mg/kg and eight mice were injectedsubcutaneously with vehicle control, phosphate buffered saline (PBS), ata volume of 100 μL per mouse. All mice were sacrificed 8 days afterfirst injection in accordance with the relevant Animal Care Guidelines.Peripheral blood (PB) samples from individual animals were collected bycardiac puncture and used for complete blood counts and differential(CBC/Diff). Femurs and spleens were harvested from each mouse.

Tests performed:

CBC/Diff Counts

PB from each mouse was collected via cardiac puncture and placed intothe appropriate microtainer tubes. Samples were sent to CLV for analysison a CellDyn 3500 counter.

Clonogenic Assays

Clonogenic progenitors of the myeloid, erythroid and lymphoid lineageswere assessed using the in vitro methylcellulose-based media systemsdescribed below.

Mature Erythroid Progenitors:

Clonogenic progenitors of the mature erythroid (CFU-E) lineages werecultured in MethoCult™ 3334, a methylcellulose-based medium containingrecombinant human (rh) Erythropoietin (3 U/mL).

Lymphoid Progenitors:

Clonogenic progenitors of the lymphoid (CFU-pre-B) lineage were culturedin MethoCult® 3630, a methylcellulose-based medium containing rhInterleukin 7 (10 ng/mL).

Myeloid and Immature Erythroid Progenitors:

Clonogenic progenitors of the granulocyte-monocyte (CFU-GM), erythroid(BFU-E) and multipotential (CFU-GEMM) lineages were cultured inMethoCult™ 3434, a methylcellulose-based medium containing recombinantmurine (rm) Stem Cell Factor (50 ng/mL), rh Interleukin 6 (10 ng/mL), rmInterleukin 3 (10 ng/mL) and rh Erythropoietin (3 U/mL).

Methods:

Mouse femurs and spleens were processed by standard protocols. Briefly,bone marrow was obtained by flushing the femoral cavity with Iscove'sModified Dulbecco's Media containing 2% fetal bovine serum (IMDM 2% FBS)using a 21 gauge needle and 1 cc syringe. Spleen cells were obtained bycrushing spleens through a 70 μM filter and rinsing the filter with IMDM2% FBS. Nucleated cell counts in 3% glacial acetic acid were thenperformed on the single cells suspensions using a Neubauer countingchamber so that the total cells per organ could be calculated. To removecontaminating red blood cells, total spleen cells were then diluted with3 times the volume of ammonium chloride lysis buffer and incubated onice 10 minutes. The cells were then washed and resuspended in IMDM 2%FBS and a second cell count were performed to determine the cellconcentration of cells after lysis.

Cell stocks were made and added to each methylcellulose-based mediaformulation to obtain the optimal plating concentrations for each tissuein each media formulation. Bone marrow cells were plated at 1×10⁵ cellsper dish in MethoCult™ 3334 to assess mature erythroid progenitors,2×10⁵ cells per dish in MethoCult™ 3630 to assess lymphoid progenitorsand 3×10⁴ cells per dish in MethoCult™ 3434 to assess immature erythroidand myeloid progenitors. Spleen cells were plated at 4×10⁵ cells perdish in MethoCult™ 3334 to assess mature erythroid progenitors, 4×10⁵cells per dish in MethoCult™ 3630 to assess lymphoid progenitors and2×10⁵ cells per dish in MethoCult™ 3434 to assess immature erythroid andmyeloid progenitors. Cultures plated in triplicate dishes were incubatedat 37° C., 5% CO2 until colony enumeration and evaluation was performedby trained personnel. Mature erythroid progenitors were cultured for 2days, lymphoid progenitors were cultured for 7 days and mature erythroidand myeloid progenitors were cultured for 12 days.

Analysis:

The mean +/−1 standard deviation was calculated for the triplicatecultures of the clonogenic assays and for the control and treatmentgroups for all data sets.

Frequency of colony forming cells (CFC) in each tissue was calculated asfollows:

Cells plated per dish

Mean CFC scored per dish

Total CFC per femur or spleen was calculated as follows:

Total CFC scored x nucleated cell count per femur or spleen (followingRBC lysis)

Number of nucleated cells cultured

Standard t-tests were performed to assess if there was a differences inthe mean number of cells or hematopoietic progenitors between the PBScontrol mice and compound treated mice. Due to the potentialsubjectivity of colony enumeration, a p value of less than 0.01 isdeemed significant. Mean values (+/−SD) for each group are shown in thetables below.

TABLE Hematologic Parameters White Blood Red Blood Treatment Cells CellsHemoglobin Hematocrit Group (×10⁹/L) (×10⁹/L) (g/L) (L/L) PBS 9.53 +/−1.44 10.5 +/− 1.1 160.9 +/− 13.3 0.552 +/− 0.057 (n = 8) ActRIIB-mFc9.77 +/− 1.19 10.8 +/− 0.3 162.1 +/− 4.1  0.567 +/− 0.019 (n = 8)

TABLE CFC From Femur and Spleen Treatment Total CFC Total CFC TotalCFU-E Total CFU-E Group per Femur per Spleen per Femur per Spleen PBS 88+/− 10 54 +/− 14 156 +/− 27 131 +/− 71  (n = 8) ActRIIB-mFc 85 +/− 9  79+/− 6* 164 +/− 23 436 +/− 86* (n = 8) *premliminary analysis indicate p< 0.05

Treatment of mice with ActRIIB(20-134)-mFc, in the brief time frame ofthis study, did not result in significant increases in red blood cell orhemoglobin content. However, the effect on progenitor cell content wasnotable. In the femurs there was no difference in the nucleated cellnumbers or progenitor content between the control and treated groups. Inthe spleens, the compound treated group experienced a statisticallysignificant increase in the nucleated cell number before red blood celllysis and in the mature erythroid progenitor (CFU-E) colony number perdish, frequency and total progenitor number per spleen. In addition, anincrease was seen in the number of myeloid (CFU-GM), immature erythroid(BFU-E) and total progenitor number per spleen. Accordingly, it isexpected that over a longer time course, ActRIIB(20-134)-mFc treatmentmay result in elevated red blood cell and hemoglobin content.

Example 8: A GDF Trap Increases Red Blood Cell Levels In Vivo

Twelve-week-old male C57BL/6NTac mice were assigned to one of twotreatment groups (N=10). Mice were dosed with either vehicle or with avariant ActRIIB polypeptide (“GDF Trap”) [ActRIIB(L79D 20-134)-hFc] bysubcutaneous injection (SC) at 10 mg/kg twice per week for 4 weeks. Atstudy termination, whole blood was collected by cardiac puncture intoEDTA containing tubes and analyzed for cell distribution using an HM2hematology analyzer (Abaxis, Inc).

Group Designation

Dose Group N Mice Injection (mg/kg) Route Frequency 1 10 C57BL/6 PBS 0SC Twice/ week 2 10 C57BL/6 GDF Trap 10 SC Twice/ [ActRIIB(L79D week20-134)-hFc]

Treatment with the GDF Trap did not have a statistically significanteffect on the number of white blood cells (WBC) compared to the vehiclecontrols. Red blood cell (RBC) numbers were increased in the treatedgroup relative to the controls (see table below). Both the hemoglobincontent (HGB) and hematocrit (HCT) were also increased due to theadditional red blood cells. The average width of the red blood cells(RDWc) was higher in the treated animals, indicating an increase in thepool of immature red blood cells. Therefore, treatment with the GDF Trapleads to increases in red blood cells, with no distinguishable effectson white blood cell populations.

Hematology Results

RBC HGB HCT RDWc 10¹²/L (g/dL) (%) (%) PBS 10.7 ± 0.1  14.8 ± 0.6  44.8± 0.4  17.0 ± 0.1  GDF Trap 12.4 ± 0.4** 17.0 ± 0.7* 48.8 ± 1.8* 18.4 ±0.2** *= p < 0.05, **= p < 0.01

Example 9: A GDF Trap is Superior to ActRIIB-Fc for Increasing Red BloodCell Levels In Vivo

Nineteen-week-old male C57BL/6NTac mice were randomly assigned to one ofthree groups. Mice were dosed with vehicle (10 mM Tris Buffered Saline,TBS), wild-type ActRIIB(20-134)-mFc, or GDF trap ActRIIB(L79D20-134)-hFc by subcutaneous injection twice per week for three weeks.Blood was collected cheek bleed at baseline and after three weeks ofdosing and analyzed for cell distribution using a hematology analyzer(HM2, Abaxis, Inc.)

Treatment with ActRIIB-Fc or the GDF trap did not have a significanteffect on white blood cell (WBC) numbers compared to vehicle controls.The red blood cell count (RBC), hematocrit (HCT), and hemoglobin levelswere all elevated in GDF Trap treated mice compared to either thecontrols or the wild-type construct (see table below). Therefore, in adirect comparison, the GDF trap promotes increases in red blood cells toa significantly greater extent than a wild-type ActRIIB-Fc protein. Infact, in this experiment, the wild-type ActRIIB-Fc protein did not causea statistically significant increase in red blood cells, suggesting thatlonger or higher dosing would be needed to reveal this effect.

Hematology Results after Three Weeks of Dosing

RBC HCT HGB (10¹²/ml) % g/dL TBS 11.06 ± 0.46 46.78 ± 1.9 15.7 ± 0.7ActRIIB-mFc 11.64 ± 0.09 49.03 ± 0.3 16.5 ± 1.5 GDF Trap  13.19 ± 0.2** 53.04 ± 0.8**  18.4 ± 0.3** **= p < 0.01

Example 10. Generation of a GDF Trap with Truncated ActRIIBExtracellular Domain

As described in Example 1, a GDF Trap referred to as ActRIIB(L79D20-134)-hFc was generated by N-terminal fusion of TPA leader to theActRIIB extracellular domain (residues 20-134 in SEQ ID NO: 1)containing a leucine-to-aspartate substitution (at residue 79 in SEQ IDNO: 1) and C-terminal fusion of human Fc domain with minimal linker(three glycine residues) (FIG. 3). A nucleotide sequence correspondingto this fusion protein is shown in FIGS. 4A and 4B.

A GDF Trap with truncated ActRIIB extracellular domain, referred to asActRIIB(L79D 25-131)-hFc, was generated by N-terminal fusion of TPAleader to truncated extracellular domain (residues 25-131 in SEQ IDNO: 1) containing a leucine-to-aspartate substitution (at residue 79 inSEQ ID NO: 1) and C-terminal fusion of human Fc domain with minimallinker (three glycine residues) (FIG. 5). A nucleotide sequencecorresponding to this fusion protein is shown in FIGS. 6A and 6B.

Example 11. Selective Ligand Binding by GDF Trap with Double-TruncatedActRIIB Extracelluar Domain

The affinity of GDF Traps and other ActRIIB-hFc proteins for severalligands was evaluated in vitro with a Biacore™ instrument. Results aresummarized in the table below. Kd values were obtained by steady-stateaffinity fit due to the very rapid association and dissociation of thecomplex, which prevented accurate determination of k_(on) and k_(off).

Ligand Selectivity of ActRIIB-hFc Variants:

Activin A Activin B GDF11 Fusion Construct (Kd e−11) (Kd e−11) (Kd e−11)ActRIIB(L79 20-134)-hFc 1.6 1.2 3.6 ActRIIB(L79D 20-134)-hFc 1350.0 78.812.3 ActRIIB(L79 25-131)-hFc 1.8 1.2 3.1 ActRIIB(L79D 25-131)-hFc 2290.062.1 7.4

The GDF Trap with a truncated extracellular domain, ActRIIB(L79D25-131)-hFc, equaled or surpassed the ligand selectivity displayed bythe longer variant, ActRIIB(L79D 20-134)-hFc, with pronounced loss ofactivin A and activin B binding and nearly full retention of GDF11binding compared to ActRIIB-hFc counterparts lacking the L79Dsubstitution. Note that truncation alone (without L79D substitution) didnot alter selectivity among the ligands displayed here [compareActRIIB(L79 25-131)-hFc with ActRIIB(L79 20-134)-hFc].

Example 12. Generation of ActRIIB(L79D 25-131)-hFc with AlternativeNucleotide Sequences

To generate ActRIIB(L79D 25-131)-hFc, the human ActRIIB extracellulardomain with an aspartate substitution at native position 79 (SEQ IDNO: 1) and with N-terminal and C-terminal truncations (residues 25-131in SEQ ID NO: 1) was fused N-terminally with a TPA leader sequenceinstead of the native ActRIIB leader and C-terminally with a human Fcdomain via a minimal linker (three glycine residues) (FIG. 5). Onenucleotide sequence encoding this fusion protein is shown in FIG. 6 (SEQID NO: 27), and an alternative nucleotide sequence encoding exactly thesame fusion protein is shown in FIGS. 9A and 9B (SEQ ID NO: 30). Thisprotein was expressed and purified using the methodology described inExample 1.

Example 13. GDF Trap with a Truncated ActRIIB Extracellular DomainIncreases Proliferation of Erythroid Progenitors in Mice

ActRIIB(L79D 25-131)-hFc was evaluated to determine its effect onproliferation of erythroid progenitors. Male C57BL/6 mice (8 weeks old)were treated with ActRIIB(L79D 25-131)-hFc (10 mg/kg, s.c.; n=6) orvehicle (TBS; n=6) on Days 1 and 4, then euthanized on Day 8 forcollection of spleens, tibias, femurs, and blood. Cells of the spleenand bone marrow were isolated, diluted in Iscove's modified Dulbecco'smedium containing 5% fetal bovine serum, suspended in specializedmethylcellulose-based medium, and cultured for either 2 or 12 days toassess levels of clonogenic progenitors at the colony-formingunit-erythroid (CFU-E) and burst forming unit-erythroid (BFU-E) stages,respectively. Methylcellulose-based medium for BFU-E determination(MethoCult M3434, Stem Cell Technologies) included recombinant murinestem cell factor, interleukin-3, and interleukin-6, which were notpresent in methylcellulose medium for CFU-E determination (MethoCultM3334, Stem Cell Technologies), while both media containederythropoietin, among other constituents. For both BFU-E and CFU-E, thenumber of colonies were determined in duplicate culture plates derivedfrom each tissue sample, and statistical analysis of the results wasbased on the number of mice per treatment group.

Spleen-derived cultures from mice treated with ActRIIB(L79D 25-131)-hFchad twice the number of CFU-E colonies as did corresponding culturesfrom control mice (P<0.05), whereas the number of BFU-E colonies did notdiffer significantly with treatment in vivo. The number of CFU-E orBFU-E colonies from bone marrow cultures also did not differsignificantly with treatment. As expected, increased numbers of CFU-Ecolonies in spleen-derived cultures were accompanied by highlysignificant (P<0.001) changes in red blood cell level (11.6% increase),hemoglobin concentration (12% increase), and hematocrit level (11.6%increase) at euthanasia in mice treated with ActRIIB(L79D 25-131)-hFccompared to controls. These results indicate that in vivo administrationof a GDF Trap with truncated ActRIIB extracellular domain can stimulateproliferation of erythroid progenitors as part of its overall effect toincrease red blood cell levels.

Example 14. GDF Trap with a Truncated ActRIIB Extracellular DomainOffsets Chemotherapy-Induced Anemia in Mice

Applicants investigated the effect of ActRIIB(L79D 25-131)-hFc onerythropoietic parameters in a mouse model of chemotherapy-inducedanemia based on paclitaxel, which inhibits cell division by blockingmicrotubule polymerization. Male C57BL/6 mice (8 weeks old) wereassigned to one of four treatments:

1) paclitaxel (25 mg/kg, i.p.)

2) ActRIIB(L79D 25-131)-hFc (10 mg/kg, i.p.)

3) paclitaxel+ActRIIB(L79D 25-131)-hFc

4) vehicle (TBS).

Paclitaxel was administered on Day 0, while ActRIIB(L79D 25-131)-hFc orvehicle were administered on Days 0 and 3. Blood samples were collectedfor CBC analysis from separate cohorts on Days 1, 3, and 5, and resultsfor treatment groups 1-3 (above) were expressed as percent differencefrom vehicle at a given time point. Attrition due to paclitaxel toxicitywas an issue in the paclitaxel-only cohort on Day 3 (where n=1);otherwise, n=3-5 per treatment per time point. Compared to vehicle,paclitaxel alone decreased hemoglobin concentration by nearly 13% at Day5, whereas addition of ActRIIB(L79D 25-131)-hFc prevented thispaclitaxel-induced decline (FIG. 11). Similar effects were observed forhematocrit and RBC levels. In the absence of paclitaxel, ActRIIB(L79D25-131)-hFc increased hemoglobin concentration by 10% compared tovehicle on Days 3 and 5 (FIG. 11). Thus, a GDF Trap with truncatedActRIIB extracellular domain can increase levels of red blood cellssufficiently to offset chemotherapy-induced anemia.

Example 15. GDF Trap with a Truncated ActRIIB Extracellular DomainReverses Nephrectomy-Induced Anemia in Mice

Applicants investigated the effect of ActRIIB(L79D 25-131)-hFc on anemiain a nephrectomized mouse model of chronic kidney disease. Male C57BL/6mice (11 weeks old) underwent either a sham operation or a unilateralnephrectomy to reduce the capacity for erythropoietin production. Micewere allowed a week for postsurgical recovery and then treatedtwice-weekly with ActRIIB(L79D 25-131)-hFc (10 mg/kg, i.p.; n=15 percondition) or vehicle (TBS; n=15 per condition) for a total of 4 weeks.Blood samples were collected before the onset of dosing and after 4weeks of treatment. Whereas vehicle-treated nephrectomized micedisplayed a significant decline in red blood cell number over the 4-weektreatment period, treatment with ActRIIB(L79D 25-131)-hFc not onlyprevented the decline but increased red blood cell levels 17% (P<0.001)above baseline (FIG. 12), despite reduced renal capacity forerythropoietin production. In nephrectomized mice, ActRIIB(L79D25-131)-hFc also generated significant increases from baseline inhemoglobin concentration and hematocrit level and, notably, stimulatedeach of these erythropoietic parameters to approximately the same extentunder nephrectomized conditions as under sham-operated conditions (FIG.13). Thus, a GDF Trap with truncated ActRIIB extracellular domain canincrease red blood cell levels sufficiently to reverse anemia in a modelof chronic kidney disease.

Example 16. GDF Trap with a Truncated ActRIIB Extracellular DomainImproves Recovery from Anemia Induced by Blood Loss in Rats

Applicants investigated the effect of ActRIIB(L79D 25-131)-hFc onerythropoietic parameters in a rat model of anemia induced by acuteblood loss (acute post-hemorrhagic anemia). Male Sprague-Dawley rats(approximately 300 g) received a chronic jugular catheter at the vendor(Harlan). On Day-1, 20% of total blood volume was withdrawn from eachrat over a 5-minute period via the catheter under isoflurane anesthesia.The volume of blood removed was based on a value for total blood volumecalculated according to the following relationship derived by Lee andco-workers (J Nucl Med 25:72-76, 1985) for rats with body weight greaterthan 120 g:Total blood volume (ml)=0.062×body weight (g)+0.0012An equal volume of phosphate-buffered saline was replaced via thecatheter at the time of blood removal. Rats were treated withActRIIB(L79D 25-131)-hFc (10 mg/kg, s.c.; n=5) or vehicle (TBS; n=5) onDays 0 and 3. Blood samples for CBC analysis were removed via thecatheter on Days-1 (baseline), 0, 2, 4, and 6.

Control rats responded to 20% blood loss with a drop of nearly 15% inred-blood-cell levels by Day 0. These levels remained significantlylower than baseline on Days 2 and 4, and had not recovered fully by Day6 (FIG. 14). Although rats treated with ActRIIB(L79D 25-131)-hFc showeda nearly identical drop in red-blood-cell levels after 20% blood loss,these rats then displayed a complete recovery in such levels by Day 2,followed by further elevation on Days 4 and 6, which represents a highlysignificant improvement over control levels at the corresponding timepoints (FIG. 14). Similar results were obtained for hemoglobinconcentration. These findings demonstrate that a GDF Trap with truncatedActRIIB extracellular domain can produce a faster recovery of red bloodcell levels from anemia caused by acute hemorrhage.

Example 17. GDF Trap with Truncated ActRIIB Extracelluar DomainIncreases Levels of Red Blood Cells in Non-Human Primates

Two GDF Traps, ActRIIB(L79D 20-134)-hFc and ActRIIB(L79D 25-131)-hFc,were evaluated for their ability to stimulate red blood cell productionin cynomolgus monkey. Monkeys were treated subcutaneously with GDF Trap(10 mg/kg; n=4 males/4 females), or vehicle (n=2 males/2 females) onDays 1 and 8. Blood samples were collected on Days 1 (pretreatmentbaseline), 3, 8, 15, 29, and 44, and were analyzed for red blood celllevels (FIG. 15), hematocrit (FIG. 16), hemoglobin levels (FIG. 17), andreticulocyte levels (FIG. 18). Vehicle-treated monkeys exhibiteddecreased levels of red blood cells, hematocrit, and hemoglobin at allpost-treatment time points, an expected effect of repeated bloodsampling. In contrast, treatment with ActRIIB(L79D 20-134)-hFc orActRIIB(L79D 25-131)-hFc increased these parameters by the firstpost-treatment time point (Day 3) and maintained them at substantiallyelevated levels for the duration of the study (FIGS. 15-17).Importantly, reticulocyte levels in monkeys treated with ActRIIB(L79D20-134)-hFc or ActRIIB(L79D 25-131)-hFc were substantially increased atDays 8, 15, and 29 compared to vehicle (FIG. 18).

This result demonstrates that GDF Trap treatment increased production ofred blood cell precursors, resulting in elevated red blood cell levels.

Taken together, these data demonstrate that truncated GDF Traps, as wellas a full-length variants, can be used as selective antagonists of GDF11and potentially related ligands to increase red blood cell formation invivo.

Example 18. GDF Trap Derived from ActRIIB5

Others have reported an alternate, soluble form of ActRIIB (designatedActRIIB5), in which exon 4, including the ActRIIB transmembrane domain,has been replaced by a different C-terminal sequence (WO2007/053775).

The sequence of native human ActRIIB5 without its leader is as follows:

(SEQ ID NO: 36) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEGPWASTTIPSGGPEATAAAGDQGSGALWLCLEGPAHE

An leucine-to-aspartate substitution, or other acidic substitutions, maybe performed at native position 79 (underlined and bolded) as describedto construct the variant ActRIIB5(L79D) which has the followingsennence:

(SEQ ID NO: 37) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNS

THLPEAGGPEGPWASTTIPSGGPEATAAAGDQGSGALWLCLEGPAHE

This variant may be connected to human Fc with a TGGG linker (SEQ ID NO:42) to generate a human ActRIIB5(L79D)-hFc fusion protein with thefollowing sequence:

(SEQ ID NO: 38) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNS

THLPEAGGPEGPWASTTIPSGGPEATAAAGDQGSGALWLCLEGPAH

VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

This construct may be expressed in CHO cells.

Example 19. Effects in Mice of Combined Treatment with EPO and a GDFTrap with a Truncated ActRIIB Extracellular Domain

EPO induces formation of red blood cells by increasing the proliferationof erythroid precursors, whereas GDF Traps could potentially affectformation of red blood cells in ways that complement or enhance EPO'seffects. Therefore, Applicants investigated the effect of combinedtreatment with EPO and ActRIIB(L79D 25-131)-hFc on erythropoieticparameters. Male C57BL/6 mice (9 weeks old) were given a single i.p.injection of recombinant human EPO alone (epoetin alfa, 1800 units/kg),ActRIIB(L79D 25-131)-hFc alone (10 mg/kg), both EPO and ActRIIB(L79D25-131)-hFc, or vehicle (Tris-buffered saline). Mice were euthanized 72h after dosing for collection of blood, spleens, and femurs.

Spleens and femurs were processed to obtain erythroid precursor cellsfor flow cytometric analysis. After removal, the spleen was minced inIscove's modified Dulbecco's medium containing 5% fetal bovine serum andmechanically dissociated by pushing through a 70-μm cell strainer withthe plunger from a sterile 1-mL syringe. Femurs were cleaned of anyresidual muscle or connective tissue and ends were trimmed to permitcollection of marrow by flushing the remaining shaft with Iscove'smodified Dulbecco's medium containing 5% fetal bovine serum through a21-gauge needle connected to a 3-mL syringe. Cell suspensions werecentrifuged (2000 rpm for 10 min) and the cell pellets resuspended inPBS containing 5% fetal bovine serum. Cells (10⁶) from each tissue wereincubated with anti-mouse IgG to block nonspecific binding, thenincubated with fluorescently labeled antibodies against mousecell-surface markers CD71 (transferrin receptor) and Ter119 (an antigenassociated with cell-surface glycophorin A), washed, and analyzed byflow cytrometry. Dead cells in the samples were excluded from analysisby counterstaining with propidium iodide. Erythroid differentiation inspleen or bone marrow was assessed by the degree of CD71 labeling, whichdecreases over the course of differentiation, and Ter119 labeling, whichis increased during terminal erythroid differentiation beginning withthe proerythroblast stage (Socolovsky et al., 2001, Blood 98:3261-3273;Ying et al., 2006, Blood 108:123-133). Thus, flow cytometry was used todetermine the number of proerythroblasts (CD71^(high)Ter119^(low)),basophilic erythroblasts (CD71^(high)Ter119^(high)),polychromatophilic+orthochromatophilic erythroblasts(CD71^(med)Ter119^(high)), and late orthochromatophilicerythroblasts+reticulocytes (CD71^(low)Ter119^(high)), as described.

Combined treatment with EPO and ActRIIB(L79D 25-131)-hFc led to asurprisingly vigorous increase in red blood cells. In the 72-h timeframe of this experiment, neither EPO nor ActRIIB(L79D 25-131)-hFc aloneincreased hematocrit significantly compared to vehicle, whereas combinedtreatment with the two agents led to a nearly 25% increase in hematocritthat was unexpectedly synergistic, i.e., greater than the sum of theirseparate effects (FIG. 19). Synergy of this type is generally consideredevidence that individual agents are acting through different cellularmechanisms. Similar results were also observed for hemoglobinconcentrations (FIG. 20) and red blood cell concentrations (FIG. 21),each of which was also increased synergistically by combined treatment.

Analysis of erythroid precursor levels revealed a more complex pattern.In the mouse, the spleen is considered the primary organ responsible forinducible (“stress”) erythropoiesis. Flow cytometric analysis of splenictissue at 72 h revealed that EPO markedly altered the erythropoieticprecursor profile compared to vehicle, increasing the number ofbasophilic erythroblasts by more than 170% at the expense of lateprecursors (late orthochromatophilic erythroblasts+reticulocytes), whichdecreased by more than one third (FIG. 22). Importantly, combinedtreatment increased basophilic erythroblasts significantly compared tovehicle, but to a lesser extent than EPO alone, while supportingundiminished maturation of late-stage precursors (FIG. 22). Thus,combined treatment with EPO and ActRIIB(L79D 25-131)-hFc increasederythropoiesis through a balanced enhancement of precursor proliferationand maturation. In contrast to spleen, the precursor cell profile inbone marrow after combined treatment did not differ appreciably fromthat after EPO alone. Applicants predict from the splenic precursorprofile that combined treatment would lead to increased reticulocytelevels and would be accompanied by sustained elevation of mature redblood cell levels, if the experiment were extended beyond 72 h.

Taken together, these findings demonstrate that a GDF Trap with atruncated ActRIIB extracellular domain can be administered incombination with EPO to synergistically increase red blood cellformation in vivo. Acting through a complementary but undefinedmechanism, a GDF trap can moderate the strong proliferative effect of anEPO receptor activator alone and still permit target levels of red bloodcells to be attained with lower doses of an EPO receptor activator,thereby avoiding potential adverse effects or other problems associatedwith higher levels of EPO receptor activation.

Example 20. GDF Trap with a Truncated ActRIIB Extracellular DomainIncreases RBC Levels in a Mouse Model of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are diverse disorders of bone marrowfailure characterized clinically by peripheral cytopenia, refractoryanemia, and risk of progression to acute myeloid leukemia. Transfusionof RBCs is a key maintenance therapy in MDS to alleviate fatigue,improve quality of life, and prolong survival; however, regulartransfusions typically result in iron overload in these patients, withadverse effects on morbidity and mortality which can lead to use ofremedies such as iron chelation therapy (Dreyfus, 2008, Blood Rev 22Suppl 2:S29-34; Jabbour et al., 2009, Oncologist 14:489-496). Althoughrecombinant erythropoietin (EPO) and its derivatives are an alternativetherapeutic approach in a small percentage of MDS patients (Estey, 2003,Curr Opin Hematol 10:60-67), recent studies suggest that this class ofagents is associated with an increased risk of morbidity and mortalityat some doses due to thromboembolic events and tumor growth (Krapf etal., 2009, Clin J Am Soc Nephrol 4:470-480; Glaspy, 2009, Annu Rev Med60:181-192). Thus, there is the need for an alternative MDS therapy thatwould increase RBC levels without the iron overload that accompanieschronic transfusions or the risks inherent in exogenous EPO and itsderivatives.

Therefore, Applicants investigated the effect of ActRIIB(L79D25-131)-hFc on RBC levels in a transgenic mouse model that recapitulatesthe key features of MDS, including transformation to acute leukemia (Linet al., 2005, Blood 106:287-295; Beachy et al., 2010, Hematol Oncol ClinNorth Am 24:361-375). Beginning at three months of age, male and femaleNUP98-HOXD13 mice were treated twice-weekly with ActRIIB(L79D25-131)-hFc (10 mg/kg, s.c.) or vehicle (TBS). Wildtype littermates weredosed with ActRIIB(L79D 25-131)-hFc or vehicle and served as controls.Blood samples were collected before the onset of dosing and at monthlyintervals thereafter to perform CBC measurements.

Several differences were noted at baseline between NUP98-HOXD13 mice andwildtype controls. Specifically, male NUP98-HOXD13 mice displayedsignificantly decreased RBC concentrations (−8.8%, p<0.05) andhematocrit (−8.4%, p<0.05) compared to wildtype mice, and femaleNUP98-HOXD13 mice showed similar trends. Results after three months ofdosing (mean±SD) are shown in the following table.

RBC Conc. Hemoglobin Conc. Hematocrit NUP98-HOXD13 Mice (10¹² cells/L)(g/dL) (%) Male Vehicle 6.56 ± 0.51 10.68 ± 0.68 31.83 ± 2.13 (n = 6)ActRIIB(L79D 25-131)-hFc    8.65 ± 0.54 ***    13.54 ± 0.91 ***    39.20± 2.82 *** (n = 8) Female Vehicle 6.38 ± 1.61 10.30 ± 2.58 31.96 ± 8.73(n = 5) ActRIIB(L79D 25-131)-hFc  8.52 ± 0.70 *  13.52 ± 0.56 *  42.23 ±2.53 † (n = 6) *** p < 0.001 vs. male + vehicle * p < 0.05 vs. female +vehicle † p = 0.056 vs. female + vehicle

Compared to vehicle, treatment with ActRIM(L79D 25-131)-hFc for threemonths increased RBC concentrations and hemoglobin concentrationssignificantly (by approximately 30%) in both male and femaleNUP98-HOXD13 mice. Hematocrit also increased significantly in these malemice and increased with a trend toward significance in the female mice.Thus, a GDF Trap with truncated ActRIIB extracellular domain canincrease RBC levels significantly in a murine model of MDS. Whereastransfusions are inherently a source of exogenous iron, a GDF Trapraises RBC levels by promoting use of endogenous iron stores viaerythropoiesis, thereby avoiding iron overloading and its negativeconsequences. In addition, as noted in Example 19, a GDF Trap actsthrough a different (albeit complementary) cellular mechanism than thatused by EPO receptor activators to stimulate erythropoiesis, and therebycircumvents potential adverse effects associated with EPO receptoractivation.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

We claim:
 1. A method for treating anemia associated with myelodysplastic syndrome in a subject in need thereof, wherein the subject is receiving or has received blood transfusions, wherein the method comprises administering to the subject an effective amount of a polypeptide comprising an amino acid sequence that is at least 90% identical to the sequence of amino acids 29-109 of SEQ ID NO: 1, wherein the polypeptide comprises an acidic amino acid at the position corresponding to position 79 of SEQ ID NO: 1, and wherein the polypeptide binds to GDF11 and/or myostatin.
 2. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of amino acids 29-109 of SEQ ID NO:
 1. 3. The method of claim 1, wherein the acidic amino acid is glutamic acid.
 4. The method of claim 1, wherein the acidic amino acid is aspartic acid.
 5. The method of claim 1, wherein the polypeptide comprises one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent.
 6. The method of claim 1, wherein the polypeptide binds to GDF11.
 7. The method of claim 1, wherein the polypeptide binds to myostatin.
 8. The method of claim 1, wherein the polypeptide binds to myostatin and GDF11.
 9. The method of claim 1, wherein the polypeptide inhibits signaling by GDF11 and myostatin in a cell-based assay.
 10. The method of claim 1, wherein the polypeptide inhibits signaling by GDF11 in a cell-based assay.
 11. The method of claim 1, wherein the polypeptide inhibits signaling by myostatin in a cell-based assay.
 12. The method of claim 1, wherein the polypeptide further comprises a constant region of an immunoglobulin.
 13. The method of claim 12, wherein the constant region is derived from an IgG heavy chain.
 14. The method of claim 13, wherein the constant region of an immunoglobulin is an Fc domain.
 15. The method of claim 14, wherein the polypeptide forms a homodimer.
 16. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:
 28. 17. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:
 28. 18. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:
 28. 19. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:
 28. 20. The method of claim 1, wherein the polypeptide comprises the sequence of amino acids 29-109 of SEQ ID NO: 1, but wherein the polypeptide comprises an acidic amino acid at the position corresponding to position 79 of SEQ ID NO:
 1. 21. The method of claim 1 wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence of amino acids 25-131 of SEQ ID NO:
 1. 22. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence of amino acids 25-131 of SEQ ID NO:
 1. 23. The method of claim 1, wherein the polypeptide comprises the sequence of amino acids 25-131 of SEQ ID NO: 1, but wherein the polypeptide comprises an acidic amino acid at the position corresponding to position 79 of SEQ ID NO:
 1. 24. The method of claim 1, wherein the subject is receiving blood transfusions.
 25. The method of claim 1, wherein the subject has refractory anemia with ringed sideroblasts. 